Water soluble paclitaxel derivatives

ABSTRACT

Disclosed are water soluble compositions of paclitaxel and docetaxel formed by conjugating the paclitaxel or docetaxel to a water soluble polymer such as poly-glutamic acid, poly-aspartic acid or poly-lysine. Also disclosed are methods of using the compositions for treatment of tumors, auto-immune disorders such as rheumatoid arthritis. Other embodiments include the coating of implantable stents for prevention of restenosis.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims priority of U.S. Ser. No. 09/050,662filed Mar. 30, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the fields ofpharmaceutical compositions to be used in the treatment of cancer,autoimmune diseases and restenosis. The present invention also relatesto the field of pharmaceutical preparations of anticancer agents such aspaclitaxel (Taxol™) and docetaxel (Taxotere), in particular makingpaclitaxel water soluble by conjugating the drug to water solublemoieties.

BACKGROUND OF THE INVENTION

[0003] Paclitaxel, an anti-microtubule agent extracted from the needlesand bark of the Pacific yew tree, Taxus brevifolia, has shown aremarkable anti-neoplastic effect in human cancer in Phase I studies andearly Phase II and III trials (Horwitz et at, 1993). This has beenreported primarily in advanced ovarian and breast cancer. Significantactivity has been documented in small-cell and non-small cell lungcancer, head and neck cancers, and in metastatic melanoma. However, amajor difficulty in the development of paclitaxel for clinical trial usehas been its insolubility in water.

[0004] Docetaxel is semisynthetically produced from 10-deacetyl baccatinIII, a noncytotoxic precursor extracted from the needles of Taxusbaccata and esterified with a chemically synthesized side chain (Cortesand Pazdur, 1995). Various cancer cell lines, including breast, lung,ovarian, and colorectal cancers and melanomas have been shown to beresponsive to docetaxel. In clinical trials, docetaxel has been used toachieve complete or partial responses in breast, ovarian, head and neckcancers, and malignant melanoma.

[0005] Paclitaxel is typically formulated as a concentrated solutioncontaining paclitaxel, 6 mg per milliliter of Cremophor EL(polyoxyethylated castor oil) and dehydrated alcohol (50% v/v) and mustbe further diluted before administration (Goldspiel, 1994). Paclitaxel(Taxol™) has shown significant activity in human cancers, includingbreast, ovarian, non-small cell lung, and head and neck cancers(Rowinsky and Donehower, 1995). It has also shown significant activityin patients with advanced breast cancer who had been treated withmultiple chemotherapeutic agents (Foa et al., 1994). As with mostchemotherapeutic agents, however, the maximum tolerated dose ofpaclitaxel is limited by toxicity. In humans, paclitaxel's major toxiceffect at doses of 100-250 mg/m² is granulocytopenia (Holmes et al.,1995); symptomatic peripheral neuropathy is its principal nonhematologictoxicity (Rowinsky et al., 1993).

[0006] The amount of Cremophor EL necessary to deliver the requireddoses of paclitaxel is significantly higher than that administered withany other drug that is formulated in Cremophor. Several toxic effectshave been attributed to Cremophor, including vasodilatation, dyspnea,and hypotension. This vehicle has also been shown to cause serioushypersensitivity in laboratory animals and humans (Weiss et al., 1990).In fact, the maximum dose of paclitaxel that can be administered to miceby i.v. bolus injection is dictated by the acute lethal toxicity of theCremophor vehicle (Eiseman et al., 1994). In addition, Cremophor EL, asurfactant, is known to leach phthalate plasticizers such asdi(2-ethylhexyl)phthalate (DEHP) from the polyvinylchloride bags andintravenous administration tubing. DEHP is known to cause hepatotoxicityin animals and is carcinogenic in rodents. This preparation ofpaclitaxel is also shown to form particulate matter over time and thusfiltration is necessary during administration (Goldspiel, 1994).Therefore, special provisions are necessary for the preparation andadministration of paclitaxel solutions to ensure safe drug delivery topatients, and these provisions inevitably lead to higher costs.

[0007] Prior attempts to obtain water soluble paclitaxel have includedthe preparation of prodrugs of paclitaxel by placing solubilizingmoieties such as succinate, sulfonic acid, amino acids, and phosphatederivatives at the 2′-hydroxyl group or at the 7-hydroxyl position(Deutsch et al., 1989; Mathew et al., Zhao and Kingston, 1991, 1992;Nicolaou et al., 1993; Vyas et al., 1995, Rose et al., 1997). While someof these prodrugs possess adequate aqueous solubility, few haveantitumor activity comparable to that of the parent drug (Deutsch etal., 1989; Mathew et al., 1992; Rose et al., 1997). Several of thesederivatives are not suitable for i.v. injection because of theirinstability in aqueous solution at neutral pH. For example, Deutsch etal. (1 989) report a 2′-succinate derivative of paclitaxel, but watersolubility of the sodium salt is only about 0.1% and the triethanolamineand N-methylglucamine salts were soluble at only about 1%. In addition,amino acid esters were reported to be unstable. Similar results werereported by Mathew et al. (1992).

[0008] Recently, Nicolaou et al. (1993) reported the synthesis and invitro biological evaluation of a novel type of prodrug termed“protaxols”. These compounds possess greater aqueous solubility and areconverted to paclitaxel as the active drug through an intramolecularhydrolysis mechanism. However, no in vivo data on the antitumor activityof protaxols are yet available. Greenwald et al. reported the synthesisof highly water-soluble 2′ and 7-polyethylene glycol esters ofpaclitaxel (Greenwald et al., 1994). Using the strategy of polymerlinkage, others have developed water-soluble polyethylene glycol(PEG)-conjugated paclitaxel (Li et al., 1996; Greenwald et al., 1996).Although these conjugates have excellent water solubility, theirtherapeutic efficacies are not better than free paclitaxel. Moreover,PEG has only two reactive functional groups at each end of its polymerchain, which effectively limit the amount of paclitaxel that PEG couldcarry (U.S. Pat. No. 5,362,831).

[0009] Other attempts to solve these problems have involvedmicroencapsulation of paclitaxel in both liposomes and nanospheres(Bartoni and Boitard, 1990). The liposome formulation was reported to beas effective as free paclitaxel, however only liposome formulationscontaining less than 2% paclitaxel were physically stable (Sharma andStraubinger, 1994). Unfortunately, the nanosphere formulation proved tobe toxic. There is still a need therefore for a water soluble paclitaxelformulation that can deliver effective amounts of paclitaxel anddocetaxel without the disadvantages caused by the insolubility of thedrug.

[0010] Another obstacle to the widespread use of paclitaxel is thelimited resources from which paclitaxel is produced, causing paclitaxeltherapy to be expensive. A course of treatment may cost several thousanddollars, for example. There is the added disadvantage that not alltumors respond to paclitaxel therapy, and this may be due to thepaclitaxel not getting into the tumor. There is an immediate need,therefore, for effective formulations of paclitaxel and related drugsthat are water soluble with long serum half lives for treatment oftumors, autoimmune diseases such as rheumatoid arthritis, as well as forthe prevention of restenosis of vessels subject to traumas such asangioplasty and stenting.

SUMMARY OF THE INVENTION

[0011] The present invention seeks to overcome these and other drawbacksinherent in the prior art by providing compositions comprising achemotherapeutic and/or antiangiogenic drug, such as paclitaxel,docetaxel, or other taxoid conjugated to a water soluble polymer such asa water soluble polyamino acid, or to a water soluble metal chelator. Itis a further embodiment of the present invention that a compositioncomprising a conjugate of paclitaxel and poly-glutamic acid hassurprising antitumor activity in animal models, and further that thiscomposition is demonstrated herein to be a new species of taxane thathas pharmaceutical properties different from that of paclitaxel. Thesecompositions are shown herein to be surprisingly effective as anti-tumoragents against exemplary tumor models, and are expected to be at leastas effective as paclitaxel, docetaxel, or other taxoid against any ofthe diseases or conditions for which taxanes or taxoids are known to beeffective. The compositions of the invention provide water solubletaxoids to overcome the drawbacks associated with the insolubility ofthe drugs themselves, and also provide the advantages of improvedefficacy and controlled release so that tumors are shown herein to beeradicated in animal models after a single intravenous administration,as well as providing a novel taxane. Poly-(l-glutamic acid) conjugatedpaclitaxel is shown in the examples hereinbelow to have a novel drugactivity, in addition to having improved the delivery to the tumor andproviding a controlled release.

[0012] The methods described herein could also be used to make watersoluble polymer conjugates of other therapeutic agents, contrast agentsand drugs, including paclitaxel, tamoxifen, Taxotere, etopside,teniposide, fludarabine, doxorubicin, daunomycin, emodin,5-fluorouracil, FUDR, estradiol, camptothecin, retinoids, verapamil,epothilones cyclosporin, and other taxoids. In particular, those agentswith a free hydroxyl group would be conjugated to the polymers bysimilar chemical reactions as described herein for paclitaxel. Suchconjugation would be well within the skill of a routine practitioner ofthe chemical art, and as such would fall within the scope of the claimedinvention. Those agents would include, but would not be limited toetopside, teniposide, camptothecin and the epothilones. As used herein,conjugated to a water soluble polymer means the covalent bonding of thedrug to the polymer or chelator.

[0013] It is also understood that the water soluble conjugates of thepresent invention may be administered in conjunction with other drugs,including other anti-tumor or anticancer drugs. Such combinations areknown in the art. The water soluble paclitaxel, docetaxel, or othertaxoid, or in preferred embodiments the poly-(l-glutamic) acidconjugated paclitaxel (PG-TXL), of the present invention may, in certaintypes of treatment, be combined with a platinum drug, an antitumor agentsuch as doxorubicin or daunorubicin, for example, or other drugs thatare used in combination with Taxol™ or combined with external orinternal irradiation, that is to say, radiation administered by anexternal radiation source, or administered systemically, for example, byinjection or ingestion of radioactive materials, such as a radioisotopecontaining formulation.

[0014] Conjugation of chemotherapeutic drugs to polymers is anattractive approach to reduce systemic toxicity and improve thetherapeutic index. Polymers with molecular mass larger than 30 kDa donot readily diffuse through normal capillaries and glomerularendothelium, thus sparing normal tissue from irrelevant drug-mediatedtoxicity (Maeda and Matsumura, 1989; Reynolds, 1995). On the other hand,it is well established that malignant tumors often have disorderedcapillary endothelium and greater permeability than normal tissuevasculature (Maeda and Matsumura, 1989; Fidler et al., 1987). Tumorsoften lack a lymphatic vasculature to remove large molecules that leakinto the tumor tissue (Maeda and Matsumura, 1989). Thus, a polymer-drugconjugate that would normally remain in the vasculature may selectivelyleak from blood vessels into tumors, resulting in tumor accumulation ofactive therapeutic drug. The water soluble polymers, such as, inpreferred embodiments PG-TXL, may have pharmacological propertiesdifferent from non-conjugated drugs (i.e. paclitaxel). Additionally,polymer-drug conjugates may act as drug depots for sustained release,producing prolonged drug exposure to tumor cells. Finally, water solublepolymers (e.g., water soluble polyamino acids) may be used to stabilizedrugs, as well as to solubilize otherwise insoluble compounds. Atpresent, a variety of synthetic and natural polymers have been examinedfor their ability to enhance tumor-specific drug delivery (Kopecek,1990, Maeda and Matsumura, 1989). However, only a few are known by thepresent inventors to be currently undergoing clinical evaluation,including SMANCS in Japan and HPMA-Dox in the United Kingdom (Maeda,1991; Kopecek and Kopeckova, 1993).

[0015] In the present disclosure, a taxoid is understood to mean thosecompounds that include paclitaxels and docetaxel, and other chemicalsthat have the taxane skeleton (Cortes and Pazdur, 1995), and may beisolated from natural sources such as the Yew tree, or from cellculture, or chemically synthesized molecules, and a preferred taxane isa chemical of the general chemical formula, C₄₇H₅₁ NO₁₄, including[2aR-[2aα,4β,4αβ,6β,9α(αR,*,βS*), 11α, 12α, 12aα,12βα,]]-β-(Benzoylamino)-α-hydroxybenzene propanoic acid 6,12b,bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca[3,4]benz-[1,2-b]oxet-9-ylester. It is understood that paclitaxel and docetaxel are each moreeffective than the other against certain types of tumors, and that inthe practice of the present invention, those tumors that are moresusceptible to a particular taxoid would be treated with that watersoluble taxoid or taxane conjugate.

[0016] In those embodiments in which the paclitaxel is conjugated to awater soluble metal chelator, the composition may further comprise achelated metal ion. The chelated metal ion of the present invention maybe an ionic form of any one of aluminum, boron, calcium, chromium,cobalt, copper, dysprosium, erbium, europium, gadolinium, gallium,germanium, holmium, indium, iridium, iron, magnesium, manganese, nickel,platinum, rhenium, rubidium, ruthenium, samarium, sodium, technetium,thallium, tin, yttrium or zinc. In certain preferred embodiments, thechelated metal ion will be a radionuclide, i.e. a radioactive isotope ofone of the listed metals. Preferred radionuclides include, but are notlimited to ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ^(99m)Tc, ⁹⁰Y, ^(114m)Sn and ^(193m)Pt.

[0017] Preferred water soluble chelators to be used in the practice ofthe present invention include, but are not limited to,diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraaceticacid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N′,N,″N″′ tetraacetate(DOTA), tetraazacyclotetradecane-N,N′,N′ ′N″′-tetraacetic acid (TETA),hydroxyethylidene diphosphonate (HEDP), dimercaptosuccinic acid (DMSA),diethylenetriaminetetramethylenephosphonic acid (DTTP) and1-(ρ-aminobenzyl)-DTPA, 1,6-diamino hexane-N,N,N′,N′-tetraacetic acid,DPDP, and ethylenebis (oxyethylenenitrilo)-tetraacetic acid, with DTPAbeing the most preferred. A preferred embodiment of the presentinvention may also be a composition comprising ¹¹¹In-DTPA paclitaxel,and Na-DTPA-paclitaxel.

[0018] In certain embodiments of the present invention, the paclitaxel,docetaxel, or other taxoid may be conjugated to a water soluble polymer,and preferably the polymer is conjugated to the 2′ or the 7- hydroxyl orboth of the paclitaxel, docetaxel, or other taxoid. Poly-glutamic acid(PG) is one polymer that offers several advantages in the presentinvention. First, it contains a large number of side chain carboxylfunctional groups for drug attachment. Second, PG can be readilydegraded by lysosomal enzymes to its nontoxic basic component,1-glutamic acid, d-glutamic acid and di-glutamic acid. Finally, sodiumglutamate has been reported to prevent manifestations of neuropathyinduced by paclitaxel, thus enabling higher doses of paclitaxel to betolerated (Boyle et al., 1996). Preferred polymers include, but are notlimited to poly(l-glutamic acid), poly(d-glutamic acid),poly(dl-glutamic acid), poly(l-aspartic acid), poly(d-aspartic acid),poly(dl-aspartic acid), poly(l-lysine), poly(d-lysine), poly(dl-lysine),copolymers of the above listed polyamino acids with polyethylene glycol,polycaprolactone, polyglycolic acid and polylactic acid, as well aspoly(2-hydroxyethyl 1-glutamine), chitosan, carboxymethyl dextran,hyaluronic acid, human serum albumin and alginic acid, withpoly-glutamic acids being particularly preferred. At the lower end ofmolecular weight, the polymers of the present invention preferably havea molecular weight of about 1,000, about 2,000, about 3,000, about4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000,about 10,000, about 11,000, about 12,000, about 13,000, about 14,000,about 15,000, about 16,000, about 17,000, about 18,000, about 19,000,about 20,000, about 21,000, about 22,000, about 23,000, about 24,000,about 25,000, about 26,000, about 27,000, about 28,000, about 29,000,about 30,000, about 31,000, about 32,000, about 33,000, about 34,000,about 35,000, about 36,000, about 37,000, about 38,000, about 39,000,about 40,000, about 41,000, about 42,000, about 43,000, about 44,000,about 45,000, about 46,000, about 47,000, about 48,000, about 49,000, toabout 50,000 kD. At the higher end of molecular weight, the polymers ofthe present invention preferably have a molecular weight of about51,000, about 52,000, about 53,000, about 54,000, about 55,000, about56,000, about 57,000, about 58,000, about 59,000, about 60,000, about61,000, about 62,000, about 63,000, about 64,000, about 65,000, about66,000, about 67,000, about 68,000, about 69,000, about 70,000, about71,000, about 72,000, about 73,000, about 74,000, about 75,000, about76,000, about 77,000, about 78,000, about 79,000, about 80,000, about81,000, about 82,000, about 83,000, about 84,000, about 85,000, about86,000, about 87,000, about 88,000, about 89,000, about 90,000, about91,000, about 92,000, about 93,000, about 94,000, about 95,000, about96,000, about 97,000, about 98,000, about 99,000, to about 100,000 kD.Within these ranges, the ranges of molecular weights for the polymersare preferably of about 5,000 to about 100,000 kD, with about 20,000 toabout 80,000 being preferred, or even about 25,000 to about 50,000 beingmore preferred.

[0019] It is a further aspect of the invention that a composition of theinvention such as PG-TXL may also be conjugated to a second lipophilicor poorly soluble antitumor agent such as camptothecin, epothilone,cisplatin, melphalan, Taxotere, etoposide, teniposide, fludarabine,verapamil, or cyclosporin, for example, or even to water soluble agentssuch as 5 fluorouracil (5 FU) or fluorodeoxyuridine (FUDR), doxorubicinor daunomycin.

[0020] It is understood that the compositions of the present inventionmay be dispersed in a pharmaceutically acceptable carrier solution asdescribed below. Such a solution would be sterile or aseptic and mayinclude water, buffers, isotonic agents or other ingredients known tothose of skill in the art that would cause no allergic or other harmfulreaction when administered to an animal or human subject. Therefore, thepresent invention may also be described as a pharmaceutical compositioncomprising a chemotherapeutic or anti-cancer drug such as paclitaxel,docetaxel, or other taxoid conjugated to a high molecular weight watersoluble polymer or to a chelator. The pharmaceutical composition mayinclude polyethylene glycol, poly-glutamic acids, poly-aspartic acids,poly-lysine, or a chelator, preferably DTPA. It is also understood thata radionuclide may be used as an anti-tumor agent, or drug, and that thepresent pharmaceutical composition may include a therapeutic amount of achelated radioactive isotope.

[0021] In certain embodiments, the present invention may be described asa method of determining the uptake of a chemotherapeutic drug such aspaclitaxel, docetaxel, or other taxoid by tumor tissue. This method maycomprise obtaining a conjugate of the drug and a metal chelator with achelated metal ion, contacting tumor tissue with the composition anddetecting the presence of the chelated metal ion in the tumor tissue.The presence of the chelated metal ion in the tumor tissue is indicativeof uptake by the tumor tissue. The chelated metal ion may be aradionuclide and the detection may be scintigraphic. The tumor tissuemay also be contained in an animal or a human subject and thecomposition would then be administered to the subject.

[0022] The present invention may also be described in certainembodiments as a method of treating cancer in a subject. This methodincludes obtaining a composition comprising a chemotherapeutic drug suchas paclitaxel, docetaxel, or other taxoid conjugated to a water solublepolymer or chelator and dispersed in a pharmaceutically acceptablesolution and administering the solution to the subject in an amounteffective to treat the tumor. Preferred compositions comprisepaclitaxel, docetaxel, or other taxoid conjugated to a water solublepolyamino acids, including but not limited to poly (l-aspartic acid),poly (daspartic acid), or poly (dl-aspartic acid), poly (l-lysine acid),poly (d-lysine acid), or poly (di-lysine acid), and more preferably topoly (1-glutamic acid), poly (d-glutamic acid, or poly (dl-glutamicacid). The compositions of the invention are understood to be effectiveagainst any type of cancer for which the unconjugated taxoid is shown tobe effective and would include, but not be limited to breast cancer,ovarian cancer, malignant melanoma, lung cancer, head and neck cancer.The compositions of the invention may also be used against gastriccancer, prostate cancer, colon cancer, leukemia, or Kaposi's Sarcoma. Asused herein the term “treating” cancer is understood as meaning anymedical management of a subject having a tumor. The term would encompassany inhibition of tumor growth or metastasis, or any attempt to inhibit,slow or abrogate tumor growth or metastasis. The method includes killinga cancer cell by non-apoptotic as well as apoptotic mechanisms of celldeath. The method of treating a tumor may include some prediction of thepaclitaxel or docetaxel uptake in the tumor prior to administering atherapeutic amount of the drug, by methods that include but are notlimited to bolus injection or infusion, as well as intraarterial,intravenous, intraperitoneal, or intratumoral administration of thedrug.

[0023] This method may include any of the imaging techniques discussedabove in which a paclitaxel-chelator-chelated metal is administered to asubject and detected in a tumor. This step provides a cost effective wayof determining that a particular tumor would not be expected to respondto DTPA-paclitaxel therapy in those cases where the drug does not getinto the tumor. It is contemplated that if an imaging technique can beused to predict the response to paclitaxel and to identify patients thatare not likely to respond, great expense and crucial time may be savedfor the patient. The assumption is that if there is no reasonable amountof chemotherapeutic agent deposited in the tumor, the probability oftumor response to that agent is relatively small.

[0024] In certain embodiments the present invention may be described asa method of obtaining a body image of a subject. The body image isobtained by administering an effective amount of a radioactive metal ionchelated to a paclitaxel-chelator conjugate to a subject and measuringthe scintigraphic signals of the radioactive metal to obtain an image.

[0025] The present invention may also be described in certain broadaspects as a method of decreasing at least one symptom of a systemicautoimmune disease comprising administering to a subject, having asystemic autoimmune disease an effective amount of a compositioncomprising paclitaxel or docetaxel conjugated to polymer, with polyaminoacids being preferred and poly-glutamic acid being more preferred. Ofparticular interest in the context of the present disclosure is thetreatment of rheumatoid arthritis, which is known to respond in somecases to paclitaxel when administered in the standard Cremophorformulation (U.S. Pat. No. 5,583,153, incorporated herein by reference).As in the treatment of tumors, it is contemplated that the effectivenessof the water soluble taxoids or taxane of the present invention will notbe diminished by the conjugation to a water soluble moiety. Therefore,the compositions of the present invention are expected to be aseffective as paclitaxel against rheumatoid arthritis. Paclitaxel is anantiangiogenic agent. Rheumatoid arthritis creates a collection of newlyformed vessels which erode the adjacent joints. It is also understoodthat the taxoid or taxane compositions of the present invention may beused in combination with other drugs, such as an angiogenesis inhibitor(AGM-1470) (Oliver et al., 1994), or other anti-cancer drugs, such asmethotrexate.

[0026] The finding that paclitaxel also inhibits restenosis afterballoon angioplasty indicates that the water soluble paclitaxels anddocetaxels of the present invention will find a variety of applicationsbeyond direct parenteral administration (WO 9625176, incorporated hereinby reference). For example, it is contemplated that water solublepaclitaxel will be useful as a coating for implanted medical devices,such as tubings, shunts, catheters, artificial implants, pins,electrical implants such as pacemakers, and especially for arterial orvenous stents, including balloon-expandable sterits. In theseembodiments it is contemplated that water soluble paclitaxel may bebound to an implantable medical device, or alternatively, the watersoluble paclitaxel may be passively adsorbed to the surface of theimplantable device. For example, stents may be coated with polymer-drugconjugates by dipping the stent in polymer-drug solution or spraying thestent with such a solution. Suitable materials for the implantabledevice should be biocompatible and nontoxic and may be chosen from themetals such as nickel-titanium alloys, steel, or biocompatible polymers,hydrogels, polyurethanes, polyethylenes, ethylenevinyl acetatecopolymers, etc. In a preferred embodiment the water soluble paclitaxel,especially a PG-TXL conjugate, is coated onto a stent for insertion intoan artery or vein following balloon angioplasty. The invention may bedescribed therefore, in certain broad aspects as a method of inhibitingarterial restenosis or arterial occlusion following vascular traumacomprising administering to a subject in need thereof, a compositioncomprising paclitaxel or docetaxel conjugated to polyglutamic acid orother water soluble poly-amino acids. In the practice of the method, thesubject may be a coronary bypass, vascular surgery, organ transplant orcoronary or any other arterial angioplasty patient, for example, and thecomposition may be administered directly, intravenously, or even coatedon a stent to be implanted at the sight of vascular trauma.

[0027] An embodiment of the invention is, therefore, an implantablemedical device, wherein the device is coated with a compositioncomprising paclitaxel or docetaxel conjugated to poly-glutamic acids orwater soluble polyamino acids in an amount effective to inhibit smoothmuscle cell proliferation. A preferred device is a stent coated with thecompositions of the present invention as described herein, and incertain preferred embodiments, the stent is adapted to be used during orafter balloon angioplasty and the coating is effective to inhibitrestenosis.

[0028] In certain preferred embodiments, the invention may be describedas a composition comprising poly-glutamic acids conjugated to the 2′ or7 hydroxyl or both of paclitaxel, docetaxel, or other taxoids, or even acomposition comprising water soluble polyamino acids conjugated to the2′ or 7 hydroxyl or both of paclitaxel, docetaxel or other taxoids.

[0029] As used herein, the terms “a poly-glutamic acid” or“poly-glutamic acids” include poly (1-glutamic acid), poly (d-glutamicacid) and poly (dl-glutamic acid), the terms “a poly-aspartic acid” or“poly-aspartic acids” include poly (l-aspartic acid), poly (d-asparticacid), poly (dl-aspartic acid), the terms “a poly-lysine” or“poly-lysine” include poly (1-lysine), poly (d-lysine), poly(dl-lysine), and the terms “a water soluble polyamino acid,” “watersoluble polyamino acids,” or “water soluble polymer of amino acids”include, but are not limited to, poly-glutamic acid, poly-aspartic acid,poly-lysine, and amino acid chains comprising mixtures of glutamic acid,aspartic acid, and/or lysine. In certain embodiments, the terms “a watersoluble polyamino acid,” “water soluble polyamino acids,” or “watersoluble polymer of amino acids” include amino acid chains comprisingcombinations of glutamic acid and/or aspartic acid and/or lysine, ofeither d and/or 1 isomer conformation. In certain preferred embodiments,such a “water soluble polyamino acid” contains one or more glutamicacid, aspartic acid, and/or lysine residues. Such “water solublepolyamino acids” may also comprise any natural, modified, or unusualamino acid described herein, as long as the majority of residues, i.e.greater than 50%, comprise glutamic acid and/or aspartic acid and/orlysine. In certain embodiments, a water soluble polymer of amino acidsthat contains more than one different type of amino acid residue issometimes referred to herein as a “co-polymer”.

[0030] In certain embodiments, various substitutions of naturallyoccurring, unusual, or chemically modified amino acids may be made inthe amino acid composition of the “water soluble polyamino acids,” andparticularly in “poly-glutamic acids,” to produce a taxoid-polyaminoacid conjugate of the present invention and still obtain moleculeshaving like or otherwise desirable characteristics of solubility and/ortherapeutic efficacy. A polyamino acid such as poly-glutamic acid,poly-aspartic acid, poly-lysine, or water soluble amino acids chain orpolymer comprising a mixture of glutamic acid, aspartic acid, and/orlysine, may, at the lower end of the amino acid substitution range, haveabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24, or about 25 or more glutamic acid, aspartic acid, or lysine,residues, respectively, substituted by any of the naturally occurring,modified, or unusual amino acids described herein. In other aspects ofthe invention, a polyamino acid such as poly-glutamic acid,poly-aspartic acid, poly-lysine, or a poly-amino acid chain comprising amixture of some or all of these three amino acids may, at the lower end,have about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21 %, about 22%, about 23%, about 24%, to about 25% or moreglutamic acid, aspartic acid, or lysine residues, respectively,substituted by any of the naturally occurring, modified, or unusualamino acids described herein.

[0031] In further aspects of the invention, a polyamino acid such aspoly-glutamic acid, poly-aspartic acid, or poly-lysine may, at the highend of the amino acid substitution range, have about 25%, about 26%,about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%,about 40%, about 41 %, about 42%, about 43%, about 44%, about 45%, about46%, about 47%, about 48%, about 49%, to about 50% or so of the glutamicacid, aspartic acid, or lysine residues, respectively, substituted byany of the naturally occurring, modified, or unusual amino acidsdescribed herein, as long as the majority of residues comprise glutamicacid and/or aspartic acid and/or lysine. In amino acid substitution ofthe various water soluble amino acid polymers, residues with ahydrophilicity index of +1 or more are preferred.

[0032] In certain aspects of the invention, the amount of anti-tumordrug conjugated per water soluble polymer can vary. At the lower end,such a composition may comprise from about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8°/a, about 9%, or about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, about 20%, about 21 % about 22%, about 23%,about 24%, to about 25% (w/w) antitumor drug relative to the mass of theconjugate. At the high end, such a composition may comprise from about26%, about 27%, about 28%, about 29%, about 30%, about 31 % about 32%,about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about39%, to about 40% or more (w/w) antitumor drug relative to the mass ofthe conjugate. Preferred anti-tumor drugs include paclitaxel, docetaxel,or other taxoids, and preferred water soluble polymers include watersoluble amino acid polymers.

[0033] In certain other aspects of the invention, the number ofmolecules of anti-tumor drug conjugated per molecule of water solublepolymer can vary. At the lower end, such a composition may comprise fromabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, to about 20 or more molecules ofantitumor drug per molecule of water soluble polymer. At the higher end,such a composition may comprise from about 21, about 22, about 23, about24, about 25, about 26, about 27, about 28, about 29, about 30, about31, about 32, about 33, about 34, about 35, about 36, about 37, about38, about 39, about 40, about 41, about 42, about 43, about 44, about45, about 46, about 47, about 48, about 49, about 50, about 51, about52, about 53, about 54, about 55, about 56, about 57, about 58, about59, about 60 about 61, about 62, about 63, about 64, about 65, about 66,about 67, about 68, about 69, about 70, about 71, about 72, about 73,about 74, to about 75 or more molecules or more of antitumor drug permolecule of water soluble polymer. Preferred anti-tumor drugs includepaclitaxel, docetaxel, or other taxoids, and preferred water solublepolymers include water soluble amino acid polymers. The preferred numberof anti-tumor drug molecules conjugated per molecule of water solublepolymer is about 7 molecules of antitumor drug per molecule of watersoluble polymer.

[0034] Water soluble amino acid polymers with various substitutions ofresidues conjugated to paclitaxel, docetaxel, or other taxoids arereferred to as “biological functional equivalents”. These “biologicallyfunctional equivalents” are part of the definition of “water solublepolyamino acids” that are conjugated to taxoids, and may be identifiedby the assays described herein as well as any applicable assay that isknown to those of skill in the art to measure improved aqueoussolubility relative to the unconjugated taxoid or taxoids used toproduce the particular water soluble amino acid polymer-taxoidcomposition. In other aspects of the invention, “biological functionalequivalents” of water soluble amino acid-taxoid polymers may be furtheridentified by improved anti-tumor cell activity, relative to theanti-tumor cell activity of the unconjugated water soluble amino acidpolymer used to produce the particular water soluble amino acidpolymer-taxoid composition by the assays described herein as well as anyapplicable assay that is known to those of skill in the art. The term“biologically functional equivalents” as used herein to describe thisaspect of the invention is further described in the detailed descriptionof the invention.

[0035] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Also as used herein,the term “a” is understood to include the meaning “one or more”.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1A. Chemical structure of paclitaxel, PEG-paclitaxel andDTPA-paclitaxel.

[0037]FIG. 1B. Chemical structure and reaction scheme for production ofPG-TXL.

[0038]FIG. 2. Effect of paclitaxel, PEG-paclitaxel and DTPA-paclitaxelon proliferation of B16 melanoma cells.

[0039]FIG. 3. Antitumor effect of DTPA-paclitaxel on MCa-4 mammarytumors.

[0040]FIG. 4. Median time (days) to reach tumor diameter of 12 mm aftertreatment with paclitaxel, DTPA-paclitaxel and PEG-paclitaxel.

[0041]FIG. 5. Gamma-scintigraphs of mice bearing MCa-4 tumors followingintravenous injection of ¹¹¹In-DTPA-paclitaxel and ¹¹¹In-DTPA. Arrowindicates the tumor.

[0042]FIG. 6. Hydrolytic degradation of PG-TXL as determined in PBS as afunction of time at different pH levels. -⋄- represents percentpaclitaxel released, -O- represents metabolite-1 produced.

[0043]FIG. 7A. Anti-tumor effect of PG-TXL against syngeneic OCA-1ovarian carcinoma tumor in female C3Hf/Kam mice. Drugs were injectedintraveneously in a single dose. Data are presented as mean±standarddeviation of tumor volumes. a, Mice bearing OCA-1 tumor were injectedwith -□-, PG control (800 mg/kg; n=9); -▴-, paclitaxel (80 mg/kg; n=7);-Δ-, paclitaxel (80 mg/kg) plus PG (800 mg/kg; n=5); --, PG-TXL (80 mgequiv. paclitaxel; n =6); or -∘-, PG-TXL (160 mg equiv. paclitaxel/kg;n=26).

[0044]FIG. 7B. Anti-tumor effect of PG-TXL against 13762F tumor infemale rats. -□- represents PG control (220 mg/kg; n=7), -▴- representspaclitaxel (20 mg/kg; n=5), -Δ- represents paclitaxel (40 mg/kg; n=7),-- represents PG-TXL (20 mg equivalent paclitaxel/kg; n=5), -∘-represents PG-TXL (40 mg or 60 mg equivalent paclitaxel/kg; n=9).

[0045]FIG. 7C. The antitumor effect of PG-TXL on mice bearing MCa-4mammary carcinoma tumors. -□- represents the response to a single i.v.dose of saline, -Δ- represents the response to a single i.v. dose of PG(0.6 g/kg); -♦- represents response to PG-TXL (40 mg/kg),-⋄- representsresponse to PG-TXL (60 mg equiv. paclitaxel/kg), -∘- represents responseto PG-TXL (120 mg/kg).

[0046]FIG. 7D. The antitumor effect of PG-TXL against soft-tissuesarcoma tumor (FSa-ll) in mice. -□- represents the response to a singlei.v. dose of saline, -⋄- represents the response to a single i.v. doseof PG (0.8 g/kg); -∘- represents response to paclitaxel (80 mg/kg), -∘-represents response to PG-TXL (160 mg equiv. Paclitaxel/kg).

[0047]FIG. 7E. The antitumor effect of PG-TXL against syngeneichepathcarcinoma tumor (HCa-I) in mice. -□- represents the response to asingle i.v. dose of saline, -Δ- represents the response to a single i.v.dose of PG (0.8 g/kg); -∘- represents response to PG-TXL (80 mg/kg), -Δ-represents response to PG-TXL (160 mg equiv. paclitaxel/kg).

[0048]FIG. 8. Release profile of paclitaxel from PEG-paclitaxel inphosphate buffer (pH 7.4). Release profiles of paclitaxel (-X-); fromPEG-paclitaxel (-∘-) at pH 7.4 is shown.

[0049]FIG. 9. Antitumor effect of PEG-paclitaxel on MCa-4 mammarytumors. -□- represents the response a single i.v. injection with asaline solution of PEG (60 mg/ml), -▪- represents the response to theCremophor/alcohol vehicle, -∘- represents a single dose of 40 mg/kg bodyweight of paclitaxel, -- represents PEG-paclitaxel at 40 mg equiv.paclitaxel/kg body weight.

[0050]FIG. 10. Tubulin polymerization assays performed at 32° C. in thepresence of 1.0 mM GTP and 1.0 mg/ml of tubulin. -□- representspaclitaxel (1.0 μM), -Δ- represents PG-TXL (10 μM equivalent paclitaxel)incubated in PBS (pH 7.4) at 37° C. for 3 days, -∘- represents freshlydissolved PG-TXL.

[0051]FIG. 11. Plasma clearance of radioactivity following an i.v.injection of PG[³H]paclitaxel and [³H]paclitaxel in C3Hf/Kam mice. -□-represents PG-TXL radioactivity after injection of 6 μCi of radiolabeledPG-[³H]paclitaxel (20 mg equivalent paclitaxel/kg), -x- representspaclitaxel radioactivity after injection of 6 μCi of radiolabeled[³H]paclitaxel (20 mg equivalent paclitaxel/kg), -∘- represents“Paclitaxel” radioactivity released from injected PG-[³H]paclitaxel.

[0052]FIG. 12A. Time-dependent OCA-1 tumor content of radioactivityfollowing injection of either PG-[³H]paclitaxel and [³H]paclitaxel intomice. Open bars represents PG-TXL radioactivity after injection of 6 μCiof radiolabeled PG-[³H]paclitaxel (20 mg equivalent paclitaxel/kg),filled bars represents paclitaxel radioactivity after injection of 6 μCiof radiolabeled [³H]paclitaxel (20 mg equivalent paclitaxel/kg).

[0053]FIG. 12B. Conversion of PG-[³H]paclitaxel to [³H]paclitaxel withinOCA-1 tumor. Total radioactivity measured after injection of 6 μCi ofradiolabeled PG-[³H]paclitaxel is shown in open bars, “Paclitaxel”derived radioactivity released from injected PG[³H]paclitaxel is shownin solid bars.

[0054]FIG. 13. Kinetics of apoptosis in OCA-1 tumors after a single i.v.dose of 160 mg equiv. paclitaxel/kg of PG-TXL (MTD) and 80 mg/kgpaclitaxel (MTD), -□- represents the response to a single i.v. dose ofPG-TXL (160 mg equiv. paclitaxel/kg MTD), -∘- represents response topaclitaxel (80 mg paclitaxel/kg MTD).

[0055]FIG. 14. Survival of nude mice with human ovarian cancer cells(SKOV3ipl) treated with PG-TXL. Five days after tumor injection, themice were injected i.v. with the PG-paclitaxel (PG-TXL), or PG control.Injections of PG-TXL were administered every seven days (▾) in the 120mg/kg group, but not the 160 mg/kg group. -▪- represents untreated mice.-▾- represents the response to multiple i.v. doses of PG. -▾- representsthe response to an i.v. dose of PG-TXL (120 mg equiv. paclitaxel/kg),-♦- represents the response to an i.v. dose of PG-TXL (160 mg equiv.paclitaxel/kg).

[0056]FIG. 15. Chemical structure and reaction scheme for production ofglutamic acid containing polyamino acids.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The present invention arises from the discovery of novel, watersoluble formulations of paclitaxel and docetaxel, and the surprisingefficacy of these formulations against tumor cells in vivo. Poly(1-glutamic acid) conjugated paclitaxel (PG-TXL) administered to micebearing ovarian carcinoma (OCA-1) caused significant tumor growth delayas compared to the same dose of paclitaxel without PG. Mice treated withpaclitaxel alone or with a combination of free paclitaxel and PG showeddelayed tumor growth initially, but tumors regrew to levels comparableto an untreated control group after ten days. Moreover, at the maximumtolerated dose (MTD) of the PG-TXL conjugate, (160 mg equiv.paclitaxel/kg), the growth of tumors was completely suppressed, thetumors shrank, and mice observed for two months following treatmentremained tumor free (MTD: defined as the maximal dose that produced 15%or less body weight loss within two wk after a single i.v. injection).In a parallel study, the antitumor activity of PG-TXL in rats with ratmammary adenocarcinoma (13762F) was examined. Again, complete tumoreradication at 40-60 mg equiv. paclitaxel/kg of PG-TXL was observed.These surprising results demonstrate that the polymer-drug conjugate,PGTXL, successfully eradicates well established solid tumors in bothmice and rats after a single intravenous injection.

[0058] In addition to the remarkable antitumor (breast, ovarian, etc.)data in syngeneic mice, good activity of PG-TXL against human breastcancer (MDA-435) and ovarian cancer (SKOV3ip1) in nude mice has recentlybeen observed. Nude mice are special animals with incomplete immunesystems in which human tumors can grow.

[0059] The data presented herein have led the present inventors toconclude that PG-TXL is a novel species of taxane that ispharmacologically distinct from previous paclitaxel or Taxol™preparations. For example, the distribution of PG-TXL within plasma isdistinct from free paclitaxel. While paclitaxel remains in the plasma ofmice for an extremely short time, PG-TXL appears to remain for a muchlonger period. This is contemplated to offer a distinct advantage inthat prolonged exposure of tumors to the drug may result in an enhancedresponse. The rate of conversion of PG-TXL to paclitaxel is slow, withless than 1% of the radioactivity from radiolabeled PG-TXL beingrecovered as radioactive paclitaxel within 48 h after injection of thepaclitaxel-polymer complex. This finding suggests that the novel drug,PG-TXL, may produce death within tumor cells in a manner which is notsimply due to the gradual release of paclitaxel itself.

[0060] Further evidence of the novelty of PG-TXL is that relatively highlevels of radioactivity from radiolabeled PG-TXL appear in tumor tissueshortly after injection. However, only small amounts of radioactivitywithin tumor tissue are due to the release of free paclitaxel.Furthermore, the percent of radioactivity within tumor tissue due topaclitaxel itself does not appreciably increase with time suggestingagain that PG-TXL is a minimal prodrug for the gradual release ofpaclitaxel. Uptake of PG-TXL versus paclitaxel has also been studied ina specialized human colon adenocarcinoma cell transport system. Whileradioactivity associated with radiolabeled PG-TXL readily gained entryinto cells, only 10% of it was due to free paclitaxel. These dataparallel that which was found in studies of tissue distribution andagain suggest that there are several mechanisms or ways in which PG-TXLmay lead to the death of cancer cells which are different from those forpaclitaxel.

[0061] In another study, it was discovered that freshly prepared PG-TXLdoes not support the growth of paclitaxel-dependent cell linessuggesting that free paclitaxel is only slowly released from thepolymer-paclitaxel complex and that the polymerpaclitaxel complex itselfis not behaving pharmacologically as “Taxol™”. Aging will promote thedegradation of PG-TXL and does increase the relative ability of theresulting material to support the growth of paclitaxel-dependent cells,but to a lesser extent than compared to free paclitaxel.

[0062] Recent analyses of tumor tissues from mice treated withpaclitaxel suggests that, as expected, this drug results in theformation of many apoptotic bodies within the tumor itself. Apoptosis isa mechanism in which cells commit self-induced death or programmed celldeath, a natural process used by an organism in wound healing and tissueremodeling. Tumors from mice treated with PG-TXL had far fewer apoptoticbodies compared to free paclitaxel but had an increased incidence oftumor necrosis and edema suggesting that paclitaxel and PG-TXL mayresult in tumor cell death by two distinctly different pathways.

[0063] These studies, and those described in the specific examples,demonstrate that PG-TXL is a new taxane which is not only extremelyactive against breast and ovarian cancers, and appears to have limitedside affects. It is now clear that the polymer conjugation of paclitaxelresults in a compound (PG-TXL) that has novel and greater over-allantitumor activity.

[0064] Another aspect of the present invention is the inclusion ofmolecules in the polymeric composition that are effective to target thetherapeutic composition to a disease or tumor site or to a particularorgan or tissue. Many of such targeting molecules are known in the artand may be conjugated to the water soluble anti-tumor compositions ofthe present invention. Examples of such molecules or agents wouldinclude, but not be limited to antibodies such as anti-tumor antibodies;anti-cell receptor antibodies; tissue specific antibodies; hormonalagents such as octreotide, estradiol and tamoxifen; growth factors; cellsurface receptor ligands; enzymes; hypoxic agents such as misonidazoleand erythronitroimidazole; and antiangiogenic agents.

[0065] Another composition of the present invention is DTPA-paclitaxel,also shown herein to be as effective as paclitaxel in an in vitroantitumor potency assay using a B16 melanoma cell line. DTPA-paclitaxeldid not show any significant difference in antitumor effect as comparedto paclitaxel against an MCa-4 mammary tumor at a dose of 40 mg/kg bodyweight in a single injection. Furthermore, ¹¹¹Indium labeledDTPA-paclitaxel was shown to accumulate in the MCa-4 tumor asdemonstrated by gammascintigraphy, demonstrating that the chelatorconjugated anti-tumor drugs of the present invention are useful andeffective for tumor imaging.

[0066] The novel compounds and methods of the present invention providesignificant advances over prior methods and compositions, as thewater-soluble paclitaxels are projected to improve the efficacy ofpaclitaxel-based anti-cancer therapy, by providing water soluble andcontrolled release paclitaxel derived compositions that also havedifferent antitumor properties than unmodified paclitaxel. Suchcompositions eliminate the need for solvents that are associated withside effects seen with prior paclitaxel compositions. In addition,radiolabeled paclitaxel, which is shown to retain anti-tumor activity,will also be useful in the imaging of tumors. Further, the presentinvention allows one to determine whether a paclitaxel will be taken upby a particular tumor by scintigraphy, single photon emission computertomography (SPECT) or positron emission tomography (PET). Thisdetermination may then be used to predict the efficacy of an anti-cancertreatment. This information may be helpful in guiding the practitionerin the selection of patients to undergo chelator-paclitaxel therapy.

[0067] The paclitaxel may be rendered water-soluble in many ways: i.e.by conjugating paclitaxel to water-soluble polymers which serve as drugcarriers, and by derivatizing the antitumor drug with water solublechelating agents. The latter approach also provides an opportunity forlabeling with radionuclides (e.g., ¹¹¹In, ⁹⁰Y, ¹⁶⁶Ho, ⁶⁸Ga, ^(99m)Tc)for nuclear imaging and/or for radiotherapy studies. The structures ofpaclitaxel, polyethylene glycol-paclitaxel (PEG-paclitaxel),poly-glutamic acid-paclitaxel conjugate (PG-TXL) anddiethylenetriaminepentaacetic acid-paclitaxel (DTPA-paclitaxel) areshown in FIG. 1.

[0068] In certain embodiments of the present invention, DTPA-paclitaxelor other paclitaxel-chelating agent conjugates, such as EDTA-paclitaxel,DTTP-paclitaxel, or DOTA-paclitaxel, for example, may be prepared in theform of water-soluble salts (sodium salt, potassium salt,tetrabutylammonium salt, calcium salt, ferric salt, etc.). These saltswill be useful as therapeutic agents for tumor treatment. Secondly,DTPA-paclitaxel or other paclitaxel-chelating agents will be useful asdiagnostic agents which when labeled with radionuclides such as ¹¹¹In or^(99m)TC, may be used as radiotracers to detect certain tumors incombination with nuclear imaging techniques. It is understood that inaddition to paclitaxel (Taxol™) and docetaxel (Taxotere), other taxanederivatives may be adapted for use in the compositions and methods ofthe present invention and that all such compositions and methods wouldbe encompassed by the present invention.

[0069] As modifications and changes may be made in the structure of thewater soluble polymer such as a water soluble polyamino acid, or a watersoluble metal chelator, of the present invention and still obtainmolecules having like or otherwise desirable characteristics, such“biologically functional equivalents” or “functional equivalents” arealso encompassed within the present invention.

[0070] For example, one of skill in the art will recognize that certainamino acids may be substituted for other amino acids in a polyamino acidstructure, including water soluble amino acid polymers such aspoly-glutamic acid, poly-aspartic acid, or poly-lysine, withoutappreciable loss of interactive binding capacity with structures suchas, for example, a chemotherapeutic and/or antiangiogenic drug, such aspaclitaxel or docetaxel, or such like. Additionally, amino acidsubstitutions in a water soluble polyamino acid conjugated to achemotherapeutic and/or antiangiogenic drug, such as paclitaxel ordocetaxel, or such like, as exemplified by but not limited to PG-TXL,may be made and still maintain part or all of the novel pharmacologicalproperties disclosed herein. Since it is the interactive capacity andnature of a protein that defines that protein's biological functionalactivity, certain amino acid sequence substitutions can be made in apolyamino acid sequence and nevertheless obtain a polyamino acid withlike (agonistic) properties. It is thus contemplated by the inventorsthat various changes may be made in the sequence of the water solublepolyamino acids of a drug conjugate, such as, but not limited to PG-TXL,without appreciable loss of their biological utility or activity.

[0071] In terms of functional equivalents, it is well understood by theskilled artisan that, inherent in the definition of a “biologicallyfunctional equivalent of a water soluble polyamino acid,” is the conceptthat there is a limit to the number of changes that may be made within aportion of the molecule and still result in a molecule with anacceptable level of equivalent biological activity. Biologicallyfunctional equivalent of a water soluble polyamino acids, are thusdefined herein as those water soluble polyamino acids in which certain,not most or all, of the amino acids may be substituted by non-watersoluble amino acids, whether natural, unusual, or chemically modified.

[0072] In particular, where shorter length water soluble polyamino acidsare concerned, it is contemplated that fewer amino acids should be madewithin the given peptide. Longer domains may have an intermediate numberof changes. The longest water soluble polyamino acid chains, asdescribed herein, will have the most tolerance for a larger number ofchanges. Of course, a plurality of distinct water soluble polyaminoacids, such as but not limited to poly glutamic acid, poly asparticacid, or poly-lysine, with different substitutions may easily be madeand used in accordance with the invention.

[0073] It is also well understood that where certain residues are shownto be particularly important to the biological or structural propertiesof a polyamino acid, such residues may not generally be exchanged. Inthis manner, functional equivalents are defined herein as those watersoluble polyamino acids which maintain a substantial amount of theirnative biological activity.

[0074] Amino acid substitutions are generally based on the relativesimilarity of the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid sidechain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

[0075] To effect more quantitative changes, the hydropathic index ofamino acids may be considered. Each amino acid has been assigned ahydropathic index on the basis of their hydrophobicity and chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

[0076] The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein, and correspondingly apolyamino acid, is generally understood in the art (Kyte & Doolittle,1982, incorporated herein by reference). It is known that certain aminoacids may be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

[0077] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. Asdetailed in U.S. Pat. No. 4,554,101, the following hydrophilicity valueshave been assigned to amino acid residues: arginine (+3.0); lysine(+3.0); aspartate (+3.0±1); glutamate (+3.0±1); senne (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). In making changes basedupon similar hydrophilicity values, the substitution of amino acidswhose hydrophilicity values are within ±2 is preferred, those which arewithin ±1 are particularly preferred, and those within ±0.5 are evenmore particularly preferred. Hence, in reference to hydrophilicity,arginine, lysine, aspartic acid, and glutamic acid are defined herein asbiologically functional equivalents, particularly in water soluble aminoacid polymers.

[0078] In addition to the water soluble polyamino acid-chemotherapeuticand/or antiangiogenic drug compounds described herein, such aspaclitaxel or docetaxel conjugated to a water soluble amino acid, orsuch like, as exemplified by, but not limited to PG-TXL compoundsdescribed herein, the inventors also contemplate that other stericallysimilar compounds may be formulated to mimic the key portions of thewater soluble polyamino acid structure. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and hence are also functional equivalents.

[0079] Certain mimetics that mimic elements of protein secondarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins, including polyamino acids, exists chiefly toorientate amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is thus designed to permit molecular interactions similar to thenatural molecule.

[0080] Some successful applications of the peptide mimetic concept havefocused on mimetics of β-turns within proteins, which are known to behighly antigenic. Likely β-turn structure within a polypeptide can bepredicted by computer-based algorithms, as discussed herein. Once thecomponent amino acids of the turn are determined, mimetics can beconstructed to achieve a similar spatial orientation of the essentialelements of the amino acid side chains.

[0081] The generation of further structural equivalents or mimetics maybe achieved by the techniques of modeling and chemical design known tothose of skill in the art. The art of receptor modeling is now wellknown, and by such methods a chemical that binds to water solublepolyamino acids can be designed and then synthesized. It will beunderstood that all such sterically designed constructs fall within thescope of the present invention.

[0082] In addition to the 20 “standard” amino acids provided through thegenetic code, modified or unusual amino acids are also contemplated foruse in the present invention. A table of exemplary, but not limiting,modified or unusual amino acids is provided herein below. TABLE 1Modified and Unusual Amino Acids Abbr. Amino Acid Abbr. Amino Acid Aad2-Aminoadipic acid EtAsn N-Ethylasparagine bAad 3-Aminoadipic acid HylHydroxylysine bAla beta-alanine, beta-Amino-propionic aHylallo-Hydroxylysine acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline4Abu 4-Aminobutyric acid, piperidinic 4Hyp 4-Hydroxyproline acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid alleallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine bAib 3-Aminoisobutyric acid Melle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornthine EtGlyN-Ethylglycine —

[0083] Toxicity studies, pharmacokinetics and tissue distribution ofDTPA-paclitaxel have shown that in mice the LD50 (50% lethal dose) ofDPTA-paclitaxel observed with a single dose intravenous (iv) injectionis about 110 mg/kg body weight. Direct comparison with paclitaxel isdifficult to make because of the dose-volume constraints imposed bylimited solubility of paclitaxel and vehicle toxicity associated with ivadministration. However, in light of the present disclosure, one skilledin the art of chemotherapy would determine the effective and maximumtolerated doses (MTD) in a clinical study for use in human subjects.

[0084] In certain embodiments of the invention, a stent coated with thepolymerpaclitaxel conjugates may be used to prevent restenosis, theclosure of arteries following balloon angioplasty. Recent results inclinical trials using balloon-expandable stents in coronary angioplastyhave shown a significant benefit in patency and the reduction ofrestenosis compared to standard balloon angioplasty (Serruys et al.,1994). According to the response-to-injury hypothesis, neointimaformation is associated with increased cell proliferation. Currently,popular opinion holds that the critical process leading to vascularlesions in both spontaneous and accelerated atherosclerosis is smoothmuscle cell (SMC) proliferation (Phillips-Hughes and Kandarpa, 1996).Since SMC phenotypic proliferation after arterial injury mimics that ofneoplastic cells, it is possible that anticancer drugs may be useful toprevent neointimal SMC accumulation. Stents coated with polymer-linkedanti-proliferative agents that are capable of releasing these agentsover a prolonged period of time with sufficient concentration will thusprevent ingrowth of hyperplastic intima and media into the lumen therebyreducing restenosis.

[0085] Because paclitaxel has been shown to suppress collagen inducedarthritis in a mouse model (Oliver et al. 1994), the formulations of thepresent invention are also contemplated to be useful in the treatment ofautoimmune and/or inflammatory diseases such as rheumatoid arthritis.Paclitaxel binding to tubulin shifts the equilibrium to stablemicrotubule polymers and makes this drug a strong inhibitor ofeukaryotic cell replication by blocking cells in the late G2 mitoticstage. Several mechanisms may be involved in arthritis suppression bypaclitaxel. For example, paclitaxel's phase specific cytotoxic effectsmay affect rapidly proliferating inflammatory cells, and furthermorepaclitaxel inhibits cell mitosis, migration, chemotaxis, intracellulartransport and neutrophil H₂O₂ production. In addition, paclitaxel mayhave antiangiogenic activity by blocking coordinated endothelial cellmigration (Oliver et al. 1994). Therefore, the water soluble polyaminoacids conjugated paclitaxel of the present invention are contemplated tobe useful in the treatment of rheumatoid arthritis. The polymerconjugated formulation disclosed herein would also offer the advantagesof controlled release of the drug and greater solubility. It is also anaspect of the treatment of arthritis that the formulations may beinjected or implanted directly into the affected joint areas.

[0086] The pharmaceutical preparations of paclitaxel or docetaxelsuitable for injectable use include sterile aqueous solutions ordispersions and sterile powders for the preparation of sterileinjectable solutions or dispersions. In all cases the form must besterile and must be fluid for injection. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier may be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The prevention of the action of microorganisms canbe brought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

[0087] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0088] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents and isotonic agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0089] The phrase “pharmaceutically acceptable” also refers to molecularentities and compositions that do not produce an allergic or similaruntoward reaction when administered to an animal or a human.

[0090] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous and intraperitoneal administration. In this connection,sterile aqueous media which can be employed will be known to those ofskill in the art in light of the present disclosure.

[0091] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE 1 Poly-Glutamic Acid-Paclitaxel (PG-TXL)

[0092] The present example concerns a first study involving theconjugation of paclitaxel to a water-soluble polymer, poly (1-glutamicacid) (PG) and the efficacy of the preparation against a variety oftumors in mice and rats. The potential of water-soluble polymers used asdrug carriers is well established (Kopecek, 1990; Maeda and Matsumura,1989).

[0093] Synthesis of Poly-Glutamic Acid-Paclitaxel (PG-TXL)

[0094] PG was selected as a carrier for paclitaxel because it can bereadily degraded by lysosomal enzymes, is stable in plasma and containssufficient functional groups for drug attachment. Several antitumordrugs, including Adriamycin (Van Heeswijk et al., 1985; Hoes et al.,1985), cyclophosphamide (Hirano et al., 1979), Ara-C (Kato et al., 1984)and melphalan (Morimoto et al., 1984) have been conjugated to PG.However, poly-aspartic acid may be conjugated to anti-tumor drugs usingthe reaction scheme described herein for PG-TXL.

[0095] The reaction scheme is presented in FIG. 1B. Poly(1-glutamicacid) (PG) sodium salt was obtained from Sigma (St. Louis, Mo.). Thepolymer by viscosity had a molecular weight of 36,200, andnumber-average molecular weight (M_(n)) of 24,000 as determined bylow-angle laser light scattering (LALLS). Lot-specific polydispersity(M_(w)/M_(n)) was 1.15 where M_(w) is weight-average molecular weight.PG sodium salt (MW 34 K, Sigma, 0.35 g) was first convened to PG in itsproton form. The pH of the aqueous PG sodium salt solution was adjustedto 2.0 using 0.2 M HCl. The precipitate was collected, dialyzed againstdistilled water, and lyophilized to yield 0.29 g PG.

[0096] To a solution of PG (75 mg, repeating unit FW 170, 0.44 mmol) indry N,N-dimethylformamide (DMF) (1.5 mL) was added 22 mg paclitaxel(0.026 mmol, molar ratio PG/paclitaxel=17), 15 mgdicyclohexyfcarbodiimide (DCC) (0.073 mmol) and trace amount ofdimethylaminopyridine (DMAP). Paclitaxel was supplied by Hande Tech(Houston, Tex.), and the purity was 99% and higher as confirmed by HPLCassay.

[0097] The reaction was allowed to proceed at room temperature for 12-18h. Thin layer chromatography (TLC, silica) showed complete conversion ofpaclitaxel (Rf=0.55) to polymer conjugate (Rf=0, CHCl3/MeOH=10:1). Tostop the reaction, the mixture was poured into chloroform. The resultingprecipitate was collected and dried in vacuum to yield 70 mgpolymer-drug conjugate. By changing the weight ratio of paclitaxel to PGin the starting materials, polymeric conjugates of various paclitaxelconcentrations can be synthesized.

[0098] The sodium salt of PG-TXL conjugate was obtained by dissolvingthe product in 1.0 M NaHCO3. The aqueous solution of PG-TXL was dialyzedagainst distilled water (MWCO 10,000) to remove low molecular weightcontaminants and excess NaHCO3 salt. Lyophilization of the dialysateyielded 98 mg of product as a white powder. The paclitaxel content inthis polymeric conjugate as determined by UV was 20-22% (w/w). Yield:98% (conversion to polymer bound paclitaxel, UV). Solubility in water>20mg paclitaxel/ml. A similar method can be used to synthesize PG-TXL withhigher paclitaxel content (up to 35%) by simply increasing the ratio ofpaclitaxel to PG used.

[0099] Characterization of Poly-Glutamic Acid-Paclitaxel (PG-TXL)

[0100] Ultraviolet spectra were obtained on a Beckman DU-70spectrophotometer, using the same concentration of PG aqueous solutionas reference. PG-TXL showed characteristic paclitaxel absorption withλ_(max) shifts from 228 to 230 nm. The concentration of paclitaxel inPG-TXL conjugate was estimated based on standard curve generated withknown concentrations of paclitaxel in methanol at absorption of 228 nmassuming that the polymer conjugate in water at 230 nm and the free drugin methanol at 228 nm have the same molar extinction and both followLambert Beer's law.

[0101]¹H-NMR spectra were recorded with GE model GN 500 (500 MHz)spectrometer in D₂O. Both the PG moieties and the paclitaxel moietieswere discernible. The couplings of polymer conjugated paclitaxel are toopoorly resolved to be measured with sufficient accuracy. Resonances at7.75 to 7.36 ppm are attributable to aromatic components of paclitaxelresonances at 6.38 ppm (C₁₀—H), 5.97 ppm (C₃—H), 5.63 ppm (C₂′—H, d),5.55-5.36 ppm (C₃′—H and C₂—H, m), 5.10 ppm (C₅—H), 4.39 ppm (C₇—H),4.10 (C₂₀—H), 1.97 ppm (OCOCH₃), and 1.18-1.20 ppm (C CH₃) aretentatively assigned to aliphatic components of paclitaxel. Otherresonances were obscured by the resonances of PG. PG resonances at 4.27ppm (H-α), 2.21 ppm (H-γ), and 2.04 ppm (H-β) are in accordance withpure PG spectrum. Although a peak at 5.63 ppm could be tentativelyassigned to the C-2′ proton of the C-2′ ester, the C-2′ proton ofunsubstituted paclitaxel at 4.78 ppm was also present, suggesting thatthe resulting conjugate may contain paclitaxel substitutions at both theC-2′ and C-7 positions. A 100 mg/ml solution of the conjugate produces aclear, viscous, yet flowable liquid. This procedure consistentlyproduces PG-TXL conjugate containing 20% of paclitaxel by weight, i.e.,approximately 7 paclitaxel molecules are bound to each polymer chain.

[0102] Gel Permeation Chromatography Studies of Poly-GlutamicAcid-Paclitaxel (PGTXL)

[0103] The relative molecular weight of PG-TXL was characterized by gelpermeation chromatography (GPC). The GPC system consisted of two LDCmodel III pumps coupled with LDC gradient master, a PL gel GPC column,and a Waters 990 photodiode array detector. The elutant (DMF) was run at1.0 ml/min with ultraviolet (UV) detection set at 270 mm. For PG-TXLsodium salt, a TSK-gel column suitable for analysis of water-solublepolymer was used, and the system was eluted with 0.2 mM PBS (pH 6.8) at1.0 ml/min. Conjugation of paclitaxel to PG resulted in an increase inthe molecular weight of PG-TXL, as indicated by the shift of retentiontime from 6.4 mm for PG to 5.0 mm for PG-TXL conjugate. The crudeproduct contained small molecular weight contaminants (retention time8.0 to 10.0 mm, and 11.3 min), which can be effectively removed byconvening PG-TXL to its sodium salt, followed by dialysis.

[0104] Hydrolytic Degradation of a Poly-Glutamic Acid-Paclitaxel(PG-TXL) Conjugate

[0105] To gain insight on the release kinetics of paclitaxel and relatedmolecular species from PG-TXL, the hydrolytic stability of PG-TXL wastested in PBS at various pH. High performance liquid chromatography(HPLC) revealed that incubation of PG-TXL in PBS solutions producedpaclitaxel and several other species including one that is morehydrophobic than paclitaxel (metabolite 1). The fact that these speciesall were derived from paclitaxel was confirmed through similardegradation studies using PG-[3H]TXL. Based on its retention time onHPLC, metabolite-1 is probably 7-epipaclitaxel, a biologically activeisomer of paclitaxel. In fact, the amount of metabolite 1 recovered inPBS surpassed that of paclitaxel after 5 days and 1 day of incubation atpH 7.4 and pH 9.5 respectively (FIG. 6). At pH 5.5 and pH 7.4, therelease profiles of metabolite 1 indicated pseudo-zero order kineticsand displayed a delay time varying from 3 days (pH 5.5) to 7 h (pH 7.4),suggesting that metabolite-1 is a secondary product. Apparently, PG-TXLis more stable in acidic solution than in basic solution.

[0106] In Vivo Antitumor Activity

[0107] All animal work was carried out at the animal facility at M.D.Anderson Cancer Center in accordance with institutional guidelines.C3H/Kam mice were bred and maintained in a pathogen-free facility in theDepartment of Experimental Radiation Oncology.

[0108] The tumor growth delay induced by PG-TXL was measured in mammaryovarian carcinoma (OCA-1) implanted in C3Hf/Kam mice. All tumors weresyngeneic to this strain. Solitary tumors were produced in the muscle ofthe right thigh of female C3H/Kam mice (25-30g) by injecting 5×10⁵murine ovarian carcinoma cells (OCA-1), mammary carcinoma (MCa-4),hepatocarcinoma (HCa-I) or fibrous sarcoma (FSa-II). In a parallelstudy, female Fischer 344 rats (125-150 g) were injected with 1.0×10⁵viable 13762F tumor cells in 0.1 ml PBS. Treatments were initiated whenthe tumors in mice had grown to 500 mm³ (10 mm in diameter), or when thetumors in rats had grown to 2400 mm³ (mean diameter 17 mm).

[0109] PG-TXL was disolved in saline (10 mg equivalent paclitaxel/ml),and paclitaxel was dissolved in Cremophor EL® vehicle (6 mg/ml). Dataare presented as mean±standard deviation of tumor volumes. In controlstudies, saline (0.6 ml), Cremophor vehicle [50/50 Cremophor/ethanoldiluted with saline (1:4)], PG solution in saline, and paclitaxel plusPG were used. The maximum tolerated dose (MTD) of PG-TXL and paclitaxelin normal female C3HtYKam mice was estimated to be 160 mg/kg and 80mg/kg respectively. A single dose of PG-TXL in saline or paclitaxel inCremophor EL vehicle was given in doses varying from 40 to 1 60 mgequiv. Paclitaxel/kg body weight. Tumor growth was determined daily(FIG. 7A, 7B, 7C, 7D and 7E) by measuring three orthogonal tumordiameters. Tumor volume was calculated according to formula (A×B×C)/2.Absolute growth delay (AGD) in mice is defined as the time in days fortumors treated with various drugs to grow from 500 to 2,000 mm³ in miceminus the time in days for tumors treated with saline control to growfrom 500 to 2,000 mm³. When the tumor size reached 2000 mm³, the tumorgrowth delay was calculated; the mice were sacrificed when tumors wereapproximately 2500 mm³. The PG-TXL group were (n=6 and 7), other eachgroup were (n=5). Table 2 summarizes acute toxicity of PG paclitaxel inrats in comparison with paclitaxel/Cremophor. Table 3 summarizes thedata concerning the effect of PG-TXL against MCa-4, FSa-II and HCa-Itumors in mice. The data are also summarized in FIG. 7A-FIG. 7E. TABLE 2Acute Toxicity of PG-TXL in Fischer Rats* # of Body Time of Full DoseToxic Weight Time at Nadir Recovery Group (mg/kg) Death Loss in % (days)(days) PG-TXL^(a) 60 1/4 15.7 7 14 PG-TXL^(a) 40 0/4 11.1 6 11Paclitaxel^(b) 60 1/4 16.7 6 15 Paclitaxel^(b) 40 0/3 17.9 6 16Paclitaxel^(b) 20 0/5 17.0 5 N/A

[0110] TABLE 3 The Antitumor Effect of PG-TXL Against Different Types ofIn vivo Murine Tumors Time to Grow^(bb) Tumor Drug^(a) 500-2000 mm³AGD^(c) t-test^(d) MCa-4 Saline 4.8 ± 0.8 (5) — — PG (0.6 g/kg) 9.3 ±1.1 (4) 4.5 0.0114 Cremophor Vehicle 6.1 ± 0.7 (5) 1.3 0.265 PG-TXL (40mg/kg) 8.6 ± 1.2 (4) 3.8 0.026 PG-TXL (60 mg/kg) 14.2 ± 1.1 (5)  9.40.0001 PG-TXL (120 mg/kg) 44.4 ± 2.9 (5)  39.6 <0.0001 Paclitaxel (40mg/kg) 9.0 ± 0.6 (4) 4.2 0.0044 Paclitaxel (60 mg/kg) 9.3 ± 0.3 (5) 4.50.0006 Fsa-II Saline 1.9 ± 0.1 (5) — — PG (0.8 g/kg) 2.8 ± 0.2 (6) 0.90.0043 Cremophor Vehicle 2.2 ± 0.2 (6) 0.3 0.122 PG-TXL (80 mg/kg) 3.8 ±0.4 (6) 1.9 0.0016 PG-TXL (160 mg/kg)  5.1 ± 0.3 (13) 3.2 <0.0001Paclitaxel (80 mg/kg) 4.2 ± 0.3 (6) 2.3 0.0002 PG + Paclitaxeql 3.0 ±0.2 (6) 1.1 0.0008 Hca-I Saline 7.3 ± 0.3 (5) — — PG (0.8 g/kg) 7.7 ±0.4 (4) 0.4 0.417 Cremophor Vehicle 6.8 ± 0.8 (5) −0.5 0.539 PG-TXL (40mg/kg) 8.2 ± 0.7 (5) 0.9 0.218 PG-TXL (80 mg/kg) 8.6 ± 0.2 (5) 1.30.0053 PG-TXL (160 mg/kg) 11.0 ± 0.8 (4)  3.7 0.0023 Paclitaxel (80mg/kg) 6.4 ± 0.5 (5) −0.9 0.138 PG + Paclitaxel 6.7 ± 0.4 (5) −0.6 0.294

[0111] Two important findings emerged from these studies. First, likepaclitaxel, there is an intertumor variability of the antitumor effectof water-soluble PG-TXL. PG-TXL is most effective against MCa-4 andOCA-1 tumors. Second, PG-TXL is more effective than paclitaxel onequivalent mg paclitaxel basis in the case of MCa-4, HCa-I, and on OCA-1tumors, and is remarkably potent at its maximum tolerated dose (MTD).

[0112] In a parallel study, the antitumor activity of PG-TXL in Fischerrats with the well established rat mammary adenocarcinoma 13762F wasexamined. Femal Fischer 344 rats (1 25-150 g) were injected with 1.0×10⁵viable 13762F tumor cells in 0.1 ml PBS. Once tumors reached a meanvolume of 2000 mm³ (mean diameter, 1.6 cm), animals were treated using asimilar protocol as described above. Tumor growth was determined dailyby measuring three orthogonal tumor diameters. Tumor volume wascalculated according to the formula (A×B×C)/2. A single dose of PG-TXLin saline or paclitaxel in a Cremophor EL® vehicle was given in dosesvarying from 20 to 60 mg equivalent paclitaxel/kg body weight. Incontrol studies, saline, the Cremophor EL® vehicle [50/50Cremophor/ethanol diluted with saline (1:4)], PG solution in saline andpaclitaxel plus PG were used. Again, complete tumor eradication at theMTD of PG-TXL (60 mg equivalent paclitaxel/kg) was observed. PG-TXLgiven at a lower dose of 40 mg equivalent paclitaxel/kg also resulted incomplete tumor regression (FIG. 7B). In contrast, the MTD of paclitaxelin Cremophor EL® was less than 20 mg/kg. Paclitaxel at this dose causeda tumor growth delay (Tumor growth delay is defined as the time in daysfor tumors treated with the test drugs to grow from 2,000 mm³ to 10,000mm³ minus the time in days for tumors treated with saline control togrow from 2,000 mm³ to 10,000 mm³) of only 5 days, whereas the sameequivalent paclitaxel dose of PG-TXL resulted in a tumor growth delay of23 days (FIG. 7B).

[0113] Studies of Nude Mice Injected with Human Breast Cancer andTreated with PGTXL

[0114] Nude mice were injected with 2×106 MDA-435-Lung2 cells (a variantof the MDA-MB-435 human breast cancer cell line) into the mammaryfatpad. When the tumors reached 5 mm mean diameter, (27 days after tumorinjection), mice were treated with an i.v. injection of PG-TXL or thevarious controls (see Table 4). Tumor measurements were taken weekly.Tumors that reached 1.5 cm were removed surgically. All mice were killedat 120 days, and remaining tumors removed and weighed. Mice wereexamined for metastases, and Lungs processed for histology, with singlesections of the organs scored for the presence of micrometastases. TABLE4 Tumor Mean tumor No. tumors Lung Treatment take^(a) wt(g)^(b)regressed^(c) metastases^(d) PBS 5/6  1.3 ± 0.24 — 4/5 (80%) Cremophor9/9 1.26 ± 0.67 — 4/8 (50%) PGA 10/10 1.13 ± 0.7  — 4/7 (57%) Taxol ®/10/10 1.31 ± 0.69 — 3/7 (42%) Cremophor 60 mg/kg 10/10 1.23 ± 0.38  2/105/8 (62.5%) PG-TXL 60 mg/kg PG-TXL 120  9/10 0.925 ± 0.12  4/8 ¼ (25%)mg/kg #160 mg/kg equivalent of PG-TXL.

[0115] From the results of the study in which a single bolus ofPG-conjugated paclitaxel (PG-TXL) was given, at a drug equivalent of 120mg/kg paclitaxel, it is apparent that the MDA-435 cancer cell lineresponds to the drug and that this formulation of the drug is muchbetter tolerated than when Cremophor is the vehicle.

[0116] In the breast cancer study using MDA-MB-435, only the higher doseof PG-TXL inhibited the growth rate of the mammary fatpad tumors. Fromthe growth curve it was apparent that tumor growth resumed approximately30 days after the single dose of conjugate. However, the growth curvedoes not reveal that in the PG-TXL 120 mg/kg group there were a numberof tumor regressions. As shown in Table 3, the incidence of lungmetastasis in the mice with residual tumors was also reduced. While thenumbers of mice in the study are small, they do suggest that the therapywas effective in reducing both local tumor growth and incidence ofmetastasis.

[0117] In this study design it is not possible to distinguish whether alower incidence of metastasis is due to a reduction of tumor mass of theprimary site, or due to a direct effect on any micrometastases that mayhave already been established at the time of therapy.

[0118] In Vivo Therapy of Human Breast Cancer Using Multiple Injectionsof PG-TXL

[0119] To test the effect of multiple injections of PG-TXL, nude micewere injected with 2×10⁶; MDA-435-Lung 2 cells (a variant of theMDA-MB-435 human breast cancer cell line) into the mammary fatpad. Whenthe tumors reached 5 mm mean diameter, the treatments were started, andrepeated at 14 day intervals (day 24, 38, 52) for a total of threeinjections. Tumor measurements were taken weekly. The mice were killedon day 105 after tumor cell injection, and the tumor weights andincidence of metastasis recorded. The lungs were processed forhistology, and single sections scored for the presence ofmicrometastases. The results are shown in Table 5. TABLE 5 Tumor TumorsTreatment take^(a) Mean weight(g)^(b) regressed^(c) Metastasis^(d) None4/5  1.83 ± 0.15 —  4/4 (100%) PG-control 6/10  1.7 ± 0.11 — 5/6 (83%)PG-TXL/60 7/10 1.36 ± 0.28 — 6/7 (86%) mg PAG- 8/10 0.97 ± 0.22 p = —2/6 (33%) TXL/120 0.011 e

[0120] Nude mice were injected i.p. with the human ovarian cancer cellline, SKOV3ipl. Five days after tumor injection, the mice were injectedi.v. with the PG-paclitaxel (PGTXL), at concentrations equivalent to 120mg/kg or 160 mg/kg of paclitaxel. Initially the plan was to repeat theseinjections at 7-day intervals, but a single injection of the 160 mg/kgdose killed 5 of the 10 mice. Only the 120 mg/kg group received threeinjections. The study was terminated on day 98, and any surviving micekilled. The results are shown in FIG. 14, and in Table 6.

[0121] The median survival values for the groups at present are:untreated=47 days, PC-control=43 days, PG-TXL (120 mg/kg)=83 days,PG-TXL (160 mg/kg)=83 days [note that this does not include the micethat died from the initial toxicity of the drug]. TABLE 6 Tumor MedianTreatment take^(a) survival (range)^(b) Ascites^(c) Mean vol (ml)^(d)None 10/10 56 (38-98) 8/10 2.2 ± 1.6 PG-control 8/9 45 (39-98) 8/8 2.2 ±1.6 PG-120 7/8 82 (59-98) 3/7 2.7 ± 1.4 PG-160  3/5^(e) 84 (34^(f)-98) 0/3

[0122] The PG-TXL 120 mg/kg significantly extended the survival of themice with intraperitoneal SKOV3ipl, (a human ovarian cancer cell linewhich overexpresses HER2/neu), compared with mice injected with PGalone. Multiple doses and/or increasing the dose of conjugate maysignificantly reduce the tumor incidence in addition to extendingsurvival.

[0123] In the nude mice studies above, the growth curves show thatalthough breast cancer growth is checked by paclitaxel, especially withthe higher dose conjugated with PG, tumor size continues to increaseabout a month after the therapy. A second (or third) round of therapymay have caused the tumor growth to plateau, or give more tumorregressions. The growth curves do not include the tumors thatregressed—as shown in Table 4, the tumors shrank/disappeared in 50% ofthe mice treated with the highest dose of PG-TXL and of the 4 animalswith progressively growing tumors at the end of the study, only one hadmicrometastases in the lungs. So the treatment that reduced growth ofthe primary tumors also reduced the incidence of metastasis. Theincidence of metastasis in all other therapy groups, including thecontrol groups of Cremophor and PG were lower than the PBS control,therefore it is probably not valid to state that the reduction inincidence of metastasis in the Taxol™/Cremophor group is a significantfinding.

EXAMPLE 2 DTPA-Paclitaxel

[0124] Synthesis of DTPA-Paclitaxel:

[0125] To a solution of pacitaxel (100 mg, 0.117 mmol) in dry DMF (2.2ml) was added diethylenetriaminepentaacetic acid anhydride (DTPA A) (210mg, 0.585 mmol) at 0° C. The reaction mixture was stirred at 4° C.overnight. The suspension was filtered (0.2 μm Millipore filter) toremove unreacted DTPA anhydride. The filtrate was-poured into distilledwater, stirred at 4° C. for 20 min, and the precipitate collected. Thecrude product was purified by preparative TLC over C₁₈ silica gel platesand developed in acetonitrile/water (1:1). Paclitaxel had an R_(f) valueof 0.34. The band above the paclitaxel with an R_(f). value of 0.65 to0.75 was removed by scraping and eluted with an acetonitrile/water (1:1)mixture, and the solvent was removed to give 15 mg of DTPA-paclitaxel asproduct (yield 10.4%): mp:>226° C. dec. The UV spectrum (sodium salt inwater) showed maximal absorption at 228 nm which is also characteristicfor paclitaxel. Mass spectrum: (FAB) m/e 1229 (M±H)⁺, 1251 (M±Na), 1267(M+K). In the ¹H NMR spectrum (DMSO-d6) the resonance of NCH₂CH₂N andCH₂COOH of DTPA appeared as a complex series of signals at δ2.71-2.96ppm, and as a multiplet at δ3.42 ppm, respectively. The resonance ofC7-H at 4.10 ppm in paclitaxel shifted to 5.51 ppm, suggestingesterification at the 7-position. The rest of the spectrum wasconsistent with the structure of paclitaxel.

[0126] The sodium salt of DTPA-paclitaxel was also obtained by adding asolution of DTPA-paclitaxel in ethanol into an equivalent amount of 0.05M NaHCO3, followed by lyophilizing to yield a water-soluble solid powder(solubility>20 mg equivalent paclitaxel/ml).

[0127] Hydrolytic Stability of DTPA-Paclitaxel:

[0128] The hydrolytic stability of DTPA-paclitaxel was studied underaccelerated conditions. Briefly, 1 mg of DTPA-paclitaxel was dissolvedin 1 ml 0.5 M NaHCO3 aqueous solution (pH 9.3) and analyzed by HPLC. TheHPLC system consisted of a Waters 150×3.9 (i.d.) mm Nova-Pak columnfilled with C18 4 μm silica gel, a Perkin-Elmer isocratic LC pump, a PENelson 900 series interface, a Spectra-Physics UV/Vis detector and adata station. The eluant (acetonitrile/methanol/0.02M ammoniumacetate=4:1:5) was run at 1.0 ml/min with UV detection at 228 nm. Theretention times of DTPA-paclitaxel and paclitaxel were 1.38 and 8.83min, respectively. Peak areas were quantitated and compared withstandard curves to determine the DTPA-paclitaxel and paclitaxelconcentrations. The estimated half-life of DTPA-paclitaxel in 0.5 MNaHCO3 solution is about 1 6 days at room temperature.

[0129] Effects of DTPA-Paclitaxel on the Growth of B16 Mouse MelanomaCells In vitro

[0130] Cells were seeded in 24-well plates at a concentration of 2.5×10⁴cells/ml and grown in a 50:50 Dulbecco's modified minimal essentialmedium (OEM) and F12 medium containing 10% bovine calf serum at 37° C.for 24 h in a 97% humidified atmosphere of 5.5% CO2. The medium was thenreplaced with fresh medium containing paclitaxel or DTPA-paclitaxel inconcentration ranging from 5×10⁻⁹ M to 75×10⁻⁹ M. After 40 h, the cellswere released by trypsinization and counted in a Coulter counter. Thefinal concentrations of DMSO (used to dissolve paclitaxel) and 0.05 Msodium bicarbonate solution (used to dissolve DTPA-paclitaxel) in thecell medium were less than 0.01%. This amount of solvent did not haveany effect on cell growth as determined by control studies.

[0131] The effects of DTPA-paclitaxel on the growth of B16 melanomacells are presented in FIG. 2. After a 40-h incubation with variousconcentrations, DTPA-paclitaxel and paclitaxel were compared as tocytotoxicity. The IC₅₀ for paclitaxel and DTPA-paclitaxel are 15 nM and7.5 nM, respectively.

[0132] Antitumor Effect on Mammary Carcinoma (MCa-4) Tumor Model:

[0133] Female C3Hf/Kam mice were inoculated with mammary carcinoma(MCa-4) in the muscles of the right thigh (5×10⁵ cells/mouse). When thetumors had grown to 8 mm (approx. 2 wks), a single dose of paclitaxel orDTPA-paclitaxel was given at 10, 20 and 40 mg equivalent paclitaxel/kgbody weight. In control studies, saline and absolute alcohol/Cremophor50/50 diluted with saline (1:4) were used. Tumor growth was determineddaily, by measuring three orthogonal tumor diameters. When the tumorsize reached 12 mm in diameter, the tumor growth delay was calculated.The mice were sacrificed when tumors were approximately 15 mm.

[0134] The tumor growth curve is shown in FIG. 3. Compared to controls,both paclitaxel and DTPA-paclitaxel showed antitumor effect at a dose of40 mg/kg. The data were also analyzed to determine the mean number ofdays for the tumor to reach 1 2 mm in diameter. Statistical analysisshowed that DTPA-paclitaxel delayed tumor growth significantly comparedto the saline treated control at a dose of 40 mg/kg (p<0.01). The meantime for the tumor to reach 12 mm in diameter was 12.1 days forDTPA-paclitaxel compared to 9.4 days for paclitaxel (FIG. 4).

[0135] Radiolabeling of DTPA-Paclitaxel with ¹¹¹In

[0136] Into a 2-ml V-vial were added successively 40 μl 0.6 M sodiumacetate (pH 5.3) buffer, 40 μl 0.06 M sodium citrate buffer (pH 5.5), 20μl DTPA-paclitaxel solution in ethanol (2% w/v) and 20 μl ¹¹¹InCl₃solution (1.0 mCi) in sodium acetate buffer (pH 5.5). After anincubation period of 30 min at room temperature, the labeled¹¹¹In-DTPA-paclitaxel was purified by passing the mixture through a CI 8Sep-Pac cartridge using saline and subsequently ethanol as the mobilephase. Free ¹¹¹In-DTPA (<3%) was removed by saline, while¹¹¹In-DTPA-paclitaxel was collected in the ethanol wash. The ethanol wasevaporated under nitrogen gas and the labeled product was reconstitutedin saline. Radiochemical yield: 84%.

[0137] Analysis of ¹¹¹In-DTPA-Paclitaxel:

[0138] HPLC was used to analyze the reaction mixture and purity of¹¹¹In-DTPA-paclitaxel. The system consisted of a LDC binary pump, a100×8.0 mm (i.d.) Waters column filled with ODS 5 μm silica gel. Thecolumn was eluted at a flow rate of 1 ml/min with a gradient mixture ofwater and methanol (gradient from 0% to 85% methanol over 15 mm). Thegradient system was monitored with a NaT crystal detector and aSpectra-Physics UVIVis detector. As evidenced by HPLC analysis,purification by Sep-Pak cartridge removed most of the ¹¹¹In-DTPA, whichhad a retention time of 2.7 min. The ¹¹¹In-DTPA was probably derivedfrom traces of DTPA contaminant in the DTPA-paclitaxel. Aradio-chromatogram of ¹¹¹In-DTPA-paclitaxel correlated with its UVchromatogram, indicating that the peak at 12.3 min was indeed the targetcompound. Under the same chromatographic conditions, paclitaxel had aretention time of 17.1 min. The radiochemical purity of the finalpreparation was 90% as determined by HPLC analysis.

[0139] Whole-Body Scintigraphy:

[0140] Female C3Hf/Kam mice were inoculated with mammary carcinoma(MCa-4) in the muscles of the right thigh (5×10⁵ cells). When the tumorshad grown to 12 mm in diameter, the mice were divided into two groups.In group 1, the mice were anesthetized by intraperitoneal injection ofsodium pentobarbital, followed by ¹¹¹In-DTPA-paclitaxel (100-200 mCi)via tail vein. A γ-camera equipped with a medium energy collimator waspositioned over the mice (3 per group). A series of 5 min acquisitionswere collected at 5, 30, 60, 120, 240 min and 24 h after injection. Ingroup 11, the same procedures were followed except that the mice wereinjected with ¹¹¹In-DTPA as a control. FIG. 5 shows gamma-scintigraphsof animals injected with ¹¹¹In-DTPA and ¹¹¹In-DTPA-paclitaxel.¹¹¹In-DTPA was characterized by rapid clearance from the plasma, rapidand high excretion in the urine with minimal retention in the kidney andnegligible retention in the tumor, the liver, the intestine and otherorgans or body parts. In contrast, ¹¹¹In-DTPA-paclitaxel exhibited apharmacological profile resembling that of paclitaxel (Eiseman et al.,1994). Radioactivity in the brain was negligible. Liver and kidney hadthe greatest tissue:plasma ratios. Hepatobmliary excretion ofradiolabeled DTPA-paclitaxel or its metabolites was one of the majorroutes for the clearance of the drug from the blood. Unlike paclitaxel,a significant amount of “In-DTPA-paclitaxel was also excreted throughkidney, which only played a minor role in the clearance of paclitaxel.The tumor had significant uptake of ¹¹¹In-DTPA-paclitaxel. These resultsdemonstrate that ¹¹¹In-DTPA-paclitaxel is able to detect certain tumorsand to quantify the uptake of ¹¹¹In-DTPA-paclitaxel in the tumors, whichin turn, may assist in the selection of patients for the paciitaxeltreatment. In contrast, the smaller PG-TXL conjugate has a differentdistribution than DTPA-paclitaxel, and partly localizes in the liver andtumors of test animals.

EXAMPLE 3 Polyethylene Glycol-Paclitaxel

[0141] Synthesis of Polyethylene Glycol-Paclitaxel (PEG-Paclitaxel)

[0142] The synthesis was accomplished in two steps. First2′-succinyl-paclitaxel was prepared according to a reported procedure(Deutsch et al., 1989). Paclitaxel (200 mg, 0.23 mmol) and succinicanhydride (288 mg, 2.22 mmol) were allowed to react in anhydrouspyridine (6 ml) at room temperature for 3 h. The pyridine was thenevaporated, and the residue was treated with water, stirred for 20 mm,and filtered. The precipitate was dissolved in acetone, water was slowlyadded, and the fine crystals were collected to yield 1 80 mg2′-succinyl-paclitaxel. PEG-paciitaxel was synthesized by anN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) mediated couplingreaction. To a solution of 2′-succinyl-paclitaxel (160 mg, 0.18 mmol)and methoxypolyoxyethylene amine (PEG-NH₂, MW 5000, 900 mg, 0.18 mmol)in methylene chloride was added EEDQ (180 mg, 0.72 mmol). The reactionmixture was stirred at room temperature for 4 h. The crude product waschromatographed on silica gel with ethyl acetate followed bychloroform-methanol (10:1). This gave 350 mg of product. ¹H NMR (CDCl₃)δ 2.76 (m, succinic acid, COCH₂CH₂CO₂), δ 3.63 (PEG, OCH₂CH₂O), δ 4.42(C7-H) and δ 5.51 (C2′-H). Maximal UV absorption was at 288 nm which isalso characteristic for paclitaxel. Attachment to PEG greatly improvedthe aqueous solubility of paclitaxel (>20 mg equivalent paclitaxel/mlwater).

[0143] Hydrolytic Stability of PEG-Paclitaxel

[0144] PEG-Paclitaxel was dissolved in phosphate buffer (0.01M) atvarious pHs at a concentration of 0.4 mM and the solutions were allowedto incubate at 37° C. with gentle shaking. At selected time intervals,aliquots (200 μl) were removed and lyophilized. The resulting drypowders were redissolved in methylene chloride for gel permeationchromatography (GPC analysis). The GPC system consisted of aPerkin-Elmer PL gel mixed bed column, a Perkin-Elmer isocratic LC pump,a PE Nelson 900 series interface, a Spectra-Physics UV/Vis detector anda data station. The elutant (methylene chloride) was run at 1.0 ml/minwith the UV detector set at 228 nm. The retention times ofPEG-paclitaxel and paclitaxel were 6.1 and 8.2 min, respectively. Peakareas were quantified and the percentage of PEG-paclitaxel remaining andthe percentage of paclitaxel released were calculated. The half life ofPEG-paclitaxel determined by linear least-squares at pH 7.4 was 54 min.The half-life at pH 9.0 was 7.6 min. Release profiles of paclitaxel fromPEG-paclitaxel at pH 7.4 is shown in FIG. 8.

[0145] Cytotoxicity Studies of PEG-Paclitaxel Using B16 Mouse MelanomaCells In Vitro

[0146] Following the procedure described in the cytotoxicity studieswith DTPA-paclitaxel melanoma cells were seeded in 24-well plates at aconcentration of 2.5×10⁴ cells/ml and grown in a 50:50 Dulbecco'smodified minimal essential medium (DME) and F12 medium containing 10%bovine calf serum at 37° C. for 24 h in a 97% humidified atmosphere of5.5% CO₂. The medium was then replaced with fresh medium containingpaclitaxel or its derivatives in concentrations ranging from 5×10⁻⁹ M to75×10⁻⁹ M. After 40 h, the cells were released by trypsinization andcounted in a Coulter counter. The final concentrations of DMSO (used todissolve paclitaxel) and 0.05 M sodium bicarbonate solution (used todissolve PEG-paclitaxel) in the cell medium were less than 0.01 %. Thisamount of solvent did not have any effect on cell growth as determinedby control studies. Furthermore, PEG in the concentration range used togenerate an equivalent paclitaxel concentration from 5×10⁻⁹ M to 75×⁻⁹ Malso did not effect cell proliferation.

[0147] Antitumor Effect of PEG-Paclitaxel Against MCa-4 Tumor in Mice

[0148] To evaluate the antitumor efficacy of PEG-paclitaxel againstsolid breast tumors, MCa-4 cells (5×10⁵ cells) were injected into theright thigh muscle of female C3Hf/Kam mice. As described in Example 1with the DTPA-paclitaxel, when the tumors were grown to 8 mm (Approx. 2wks), a single dose of paclitaxel or PEG-paclitaxel was given at 10, 20and at 40 mg equivalent paclitaxel/kg body weight. Paclitaxel wasinitially dissolved in absolute ethanol with an equal volume ofCremophor. This stock solution was further diluted (1:4 by volume) witha sterile physiological solution within 1 5 min of injection.PEG-paclitaxel was dissolved in saline (6 mg equiv. paclitaxel/ml) andfiltered through a sterile filter (Millipore, 4.5 μm). Saline,paclitaxel vehicle, absolute alcohol:Cremophor (1:1) diluted with saline(1:4) and PEG solution in saline (600 mg/kg body weight) were used incontrol studies. Tumor growth was determined daily, by measuring threeorthogonal tumor diameters. When the tumor size reached 1 2 mm indiameter, the tumor growth delay was calculated.

[0149] The tumor growth curve is shown in FIG. 9. At a dose of 40 mg/kg,both PEG-paclitaxel and paclitaxel effectively delayed tumor growth.Paclitaxel was more effective than PEG-paclitaxel, although thedifference was not statistically significant. Paclitaxel treated tumorsrequired 9.4 days to reach 12 mm in diameter whereasPEG-paclitaxel-treated tumors required 8.5 days. Statistically, thesevalues were significant (p>0.05) as compared to their correspondingcontrols, which were 6.7 days for the paclitaxel vehicle and 6.5 daysfor the saline solution of PEG (FIG. 4).

EXAMPLE 5 Poly(L-glutamic acid)-Paclitaxel (PG-TXL) and PaclitaxelPharmacological Properties.

[0150] The objective of this study was to compare PG-TXL and paclitaxelpharmacological properties in their ability to promote in vitro assemblyof tubulin, to inhibit cell growth against rat mammary tumor cell line13762F and human breast tumor cell lines, to induce p53 protein, and torescue a paclitaxel-dependent mutant cell line. Paclitaxel's releasefrom PG-TXL in vivo was measured to determine if PG-TXL's mechanism ofaction can be attributed to free pacitaxel.

[0151] Microtubule Polymerization Using Poly-Glutamic Acid-Paclitaxel(PG-TXL) and Paclitaxel

[0152] To test whether intact PG-TXL has any intrinsic biologicalactivity in promoting tubulin polymerization, paclitaxel, PG-TXL, and“aged” PG-TXL were compared for their relative ability to promote invitro assembly of purified bovine brain tubulin. The tubulin assemblyreaction was performed at 32° C. in PEM buffer (80 mM PIPES buffer, 1 mMEGTA, 1 mM MgCl₂, pH 6.9) at a tubulin (bovine brain, Cytoskeleton Inc.,Boulder, Colo.) concentration of 1 mg/ml (10 μM) in the presence orabsence of drugs (1.0 μM equivalent paclitaxel) and 1.0 mM guanosine5′-triphosphate (GTP). “Aged” PG-TXL was obtained by placing PG-TXL inPBS (pH 7.4) at 37° C. for 3 days. Tubulin polymerization was followedby measuring the absorbance of the solution at 340 nm over time.Addition of 1 μM paclitaxel to a solution of tubulin in assembly buffercaused a clear increase in absorbance due to the increase in lightscattering resulting from the polymerization of tubulin intomicrotubules. In contrast, a 10 μM paclitaxel equivalent of PG-TXL hadno effect on polymerization. A solution of the conjugate that was “aged”for 3 days in PBS (pH 7.4) at 37° C. exhibited enhanced activityalthough its ability to promote tubulin polymerization was stillmarkedly less than paclitaxel (FIG. 10).

[0153] Effects of Poly-Glutamic Acid-Paclitaxel (PG-TXL) on the Growthof Rat and Human Tumor Cell Lines In Vitro

[0154] To evaluate whether the superior antitumor activity of PG-TXLobserved in animals is due to increased cytotoxicity, PG-TXL andpaclitaxel were compared for their ability to inhibit cell growthagainst the established rat mammary tumor cell line 13762F. The effectof PG-TXL on cell growth was examined by a plating efficiency assay. Rat13762F cells were seeded (200 cells) into 60 mm dishes containing drugconcentrations varying from 0 to 200 nM in growth medium (α modifiedminimum essential medium [α-MEM] containing 5% fetal bovine serum, 50U/ml of penicillin, and 50 μg/ml of streptomycin). After 6 days ofgrowth, the cells were stained with a 0.1 % methylene blue solution andcolonies were counted. The drug concentration producing 50% inhibitionof colony formation (IC₅₀) was then calculated. The approximate IC₅₀values after 6 days of continuous exposure were: paclitaxel (2 nM),PG-TXL (100 nM), “aged” (see below) PG-TXL (50 nM). It is clear thatPG-TXL is approximately 30-50 fold less potent than paclitaxel itself.When PG-TXL was incubated in phosphate buffered saline solution (PBS, pH74) at 37° C. for 3 days to obtain an “aged” solution, only about 10% ofpaclitaxel was released. Since the “aged” solution is more potent thanfreshly dissolved PG-TXL, the in vitro degradation of PG-TXL or releaseof active drug appears to be important for PG-TXL to exert thisbiological activity. However, even after “aging,” PG-TXL is still 25times less potent than paclitaxel.

[0155] In a similar study, the effect of PG-TXL on cell growth of humanbreast cancer cell lines was examined by MTT assay after 3 days ofcontinuous exposure. While PG-TXL was 8-and 30-fold more potent thanpaclitaxel against MDA330 and MDA-MB453 cell lines, PG-TXL was 2- and3-fold less potent than paclitaxel against MCF7/her-2 and MCF7 celllines. These results suggest that PG-TXL and paclitaxel have differentactivity against different cell lines. PG-TXL may be a product withdistinct pharmacological properties different from that of paclitaxel.

[0156] The Ability of Poly-Glutamic Acid-Paclitaxel (PG-TXL) to Supporta Paclitaxel-Dependent Cell Line In Vitro

[0157] The inventors investigated the ability of PG-TXL to rescue apaclitaxel-dependent mutant cell line. Tax 18, a CHO cell line selectedfor resistance to paclitaxel, is a well characterized mutant that hasbeen found to require the continuous presence of paclitaxel for celldivision. In the absence of drug, a functional mitotic spindle apparatusis unable to form (Cabral et al. 1983). The mitosis phase of the cellcycle is prolonged with subsequent failure to segregate chromosomes andto divide into daughter cells. Nonetheless, the cells continue toprogress through the cell cycle and replicate their DNA resulting in theformation of large polyploid cells that eventually die due to genomicinstability (Cabras and Barlow, 1991). Low concentrations of paclitaxelare able to rescue the mutant phenotype by permitting microtubuleassembly and the formation of sufficient mitotic spindle fibers. Thus,these cells provide a convenient bioassay-for agents that promotemicrotubule assembly. Growth of paclitaxel-dependent CHO mutant Tax-18cells was carried out on 24-well tissue culture dishes. Approximately100 cells were added to wells containing growth medium and equivalentconcentrations of paclitaxel varying from 0 to 1.0 μM. After 6 days ofincubation at 37° C., the medium was removed and the cells were stainedwith methylene blue.

[0158] Little or no increase in cell number is seen in the absence ofdrug, but concentrations of paclitaxel between 0.05-0.2 μM clearlysupport the growth of this cell line. Higher concentrations ofpaclitaxel are presumably toxic to the cells because ofoverstabilization of the microtubules as is observed for normal cells.On the other hand, freshly prepared PG-TXL shows little ability torescue Tax-18 cell growth even at the highest paclitaxel-equivalentconcentration tested (1 μM). When PG-TXL was “aged” by incubating in PBSfor 6 days at 37° C., its ability to support Tax-18 cell growth waspartially restored. These data indicate that PG-TXL does not promotemicrotubule assembly, and that the in vitro biological activity of“aged” PG-TXL is a contribution of paclitaxel released frompoly-glutamic acid-paclitaxel (PG-TXL).

[0159] The Release of [³H]paclitaxel from PG-[³H]TXL In Vivo

[0160] To assess the pharmacokinetic and release characteristics ofpaclitaxel in vivo, normal female C3Hf/Kam mice (25-30 g) were injectedwith a dose of 20 mg equivalent [³H]paclitaxel or PG-[³H]paclitaxelintravenously into the tail vein. Each mouse received 6 μCi ofradiolabeled drug. [³H]paclitaxel was dissolved in Cremophor EL® vehiclewhereas PG-[³H]paclitaxel was dissolved in saline. Volume injected intoeach mice was between 0.2 to 0.3 ml. At 0, 5, 15, 30 min, and 1, 2, 4,8, 16, 24, 48 h postinjection, animals were sacrificed and blood sampleswere collected (4-5 mice per time point). Total radioactivity in plasmawas measured by liquid scintillation counting (Beckman Model LS 6500,Fullerton, Calif.) using 10 μl aliquots of plasma. Up to 200 μl plasmawas extracted with 3 volume of ethyl acetate according to Longnecker etal. (1987). The extraction efficiency for paclitaxel was 80%. Thesamples were centrifuged for 5 min at 2500 rpm, and the supernatant wasseparated and brought to dryness. The dried extract was reconstitutedwith 195 μl of HPLC mobile phase, mixed with 5 μl of cold paclitaxel(0.2 mg/ml), and 100 μl was injected onto the HPLC for determination offree paclitaxel radioactivity. Pharmacokinetic parameters were analyzedby a noncompartmental model using the WinNonlin software package. Eachdata point generated was the mean value of 4-5 mice.

[0161] The clearance of both drugs from plasma is shown in FIG. 11.While paclitaxel has an extremely short half life in plasma of mice(t_(1/2), 29 min), the apparent half life of PG-TXL is prolonged(t_(1/2), 317 min). Slower clearance of PG-TXL from the blood was adesign feature of the polymer-drug conjugate with the goal of improvingtumor uptake. Surprisingly, the rate of conversion of PG-TXL topaclitaxel in plasma is slow with less than 0.1% of the radioactivityfrom PG-[³H]TXL being recovered as [³H]paclitaxel within 144 h afterdrug injection (FIG. 11).

[0162] In a separate study, mice bearing OCA-1 tumors were prepared asdescribed previously. When the tumor reached 500 mm³, animals wereinjected with a dose of 20 mg equivalent paclitaxel/kg of [³H]paclitaxelor PG-[³H]TXL into the tail vein. Animals were killed at 2, 5, 9, 24,48, and 144 h postinjection. Tumors were removed, weighed, andhomogenized with 3 volume of PBS (w/v). Percent of injected dose pergram tissue is calculated based on total radioactivity associated withthe tumor, which was determined by counting prepared tissue homogenatealiquots. An aliquot of tissue homogenate was mixed with tissuesolubilizer, followed by addition of scintillation solvent, and countedfor total radioactivity. The counting efficiency was verified by themethod of standard addition. Alternatively, aliquots of tissuehomogenates were extracted with ethyl acetate and analyzed for freepaclitaxel by HPLC. The HPLC system consisted of a 150×3.9 mm Nova-Pakcolumn (Waters, Milford, Mass.), a liquid chromatography pump (Watersmodel 510), a UV/Vis detector set at 228 nm (Waters model 486), a flowscintillation analyzer (Packard model 500TR, Downers Grove, Ill.), and aPackard radiomatic software for data analysis. The eluting solvent(methanol:watter=2:1) was run at 1.0 ml/min. The uptake of total drugsin OCA-1 tumor was expressed as a percentage of the administered doseper gram of tissue and the association of radioactivity within OCA-1tumor as free paclitaxel was expressed as dpm per gram tissue.

[0163] Quantitative assessment of tumor uptake in C3Hf/Kam mice showedthat relatively high levels of radioactivity from radiolabeled PG-TXLappear in tumor tissue shortly after injection (FIG. 12A) as compared toradiolabeled paclitaxel. However, only small amounts of radioactivitywithin tumor tissue are due to the release of free paclitaxel (FIG.12B). Data are presented in FIG. 12A and FIG. 12B as mean±SD from 3 miceper time point. The percent of radioactivity within tumor tissue due topaclitaxel does not appreciably increase with time suggesting thatPG-TXL is not simply a prodrug for the gradual release of paclitaxel.

[0164] In contrast to paclitaxel, in vitro studies with PG-TXL whetherprepared as a fresh solution or even after “aging” in buffer haveclearly shown that the complex is not a potent cytotoxic species. Itneither strongly supports tubulin polymerization nor the growth andsurvival of a paclitaxel-dependent CHO cell line. Furthermore, dataobtained from pharmacokinetic studies indicate that both the extent andrate of release of paclitaxel in plasma is very low (less than 0.1% in144 h). While the uptake of PG-TXL material was some 5-fold greater thanthat achieved by paclitaxel when using equivalent antitumor doses, thatmaterial which gains entry into tissues exists in the tissue mainly inform(s) which have been shown not to be free paclitaxel.

EXAMPLE 6 Effect of Polymer Structure on Activity of Water solublepolyamino Acid-Paclitaxel Conjugates.

[0165] The present study evaluated whether antitumor activities ofpolymer-paclitaxel conjugates were affected by the structure ofpolyamino acids used for drug conjugation. Paclitaxel was coupled topoly(l-glutamic acid), poly(d-glutamic acid), and poly(laspartic acid)according to previously described procedures. These polyaminoacidpaclitaxel conjugates had similar paclitaxel content, aqueoussolubility, and molecular weight (30-40K). In C3Hf/Kam mice bearingmurine OCA-1 ovarian cancer (500 mm³ at time of treatment), a singlei.v. injection of poly(l-glutamic acid)-paclitaxel at 80 mg equiv.paclitaxel/kg body weight produced a tumor growth delay of 21 days vs.saline treated controls. Poly(d-glutamic acid)-paclitaxel was aseffective as poly(l-glutamic acid)-paclitaxel. However, paclitaxelconjugated with poly(l-aspartic acid) was completely inactive againstOCA-1 tumor. In a separate study, the antitumor activity ofpolymer-paclitaxel conjugates of different molecular weight (1K, 13K,and 36K) was compared. Conjugates of lower molecular weight weresignificantly less effective than conjugate of higher molecular weight.The higher molecular weights above 50,000 was too viscous.

EXAMPLE 7 Poly-glutamic Acid-Paclitaxel (PG-TXL) Induces less ApoptosisCompared to Paclitaxel.

[0166] To assess the mechanism of PG-TXL associated antitumor activity,histological sections of OCA-1 tumors excised from paclitaxel and PG-TXLtreated mice were examined. OCA-1 tumor bearing mice were prepared aspreviously described. When tumor volume reached 500 mm³, animals wereinjected with either paclitaxel (80 mg/kg) or PG-TXL (160 mg equivalentpaclitaxel/kg). At different times ranging from 0 to 144 h aftertreatment, tumors were histologically analyzed to quantify mitotic andapoptotic activity according to Milas et al. (1995). The mice werekilled by cervical dislocation and the tumors were immediately excisedand placed in neutral-buffered formalin. The tissues were then processedand stained with hematoxylin and eosin. Both mitosis and apoptosis werescored in coded slides by microscopic examination at 400× magnification.Five fields of nonnecrotic areas were randomly selected in eachhistological specimen, and in each field the number of apoptotic nucleiand cells in mitosis were recorded as numbers per 100 nuclei and wereexpressed as a percentage. The values were based on scoring 1500 nucleiobtained from 3 mice per time point.

[0167] The changes observed in the paclitaxel-treated mice werequalitatively similar to those previously described (Milas et al.,1995). The tumor cells showed marked nuclear fragmentation withformation of apoptotic bodies, which was especially marked on day 1(FIG. 13). Viable tumor cell clumps with normal mitoses were stillpresent in these tumors by 144 h, indicating that these tumors wouldeventually regrow. Treatment with PG-TXL only resulted in a mildincrease in mitotically arrested cells and apoptotic cells, presumablydue to the small amount of free paclitaxel released from PG-TXL (FIG.13). By 96 h, tumors from PG-TXL-treated mice developed extensive edemaand necrosis, and only a small rim of viable tumor cells remained. By144 h, the residual tumor clumps as compared to controls were comprisedof cells that were larger, more pleomorphic, and that displayed lessmitotic activity.

[0168] These data suggest that the water-soluble PG-TXL conjugate of thepresent disclosure has superior antitumor efficacy with reduced toxicityas compared to conventional free paclitaxel preparations. Althoughoriginally designed as a water-soluble form of paclitaxel, it is nowclear that the agent used to solubilize paclitaxel, contributes to theoverall anti-tumor activity of this remarkable new complex. These dataindicate that PG-TXL has an ability to produce cell death in a mannerwhich is separate from and in addition to the apoptosis produced byreleased free paclitaxel.

EXAMPLE 8 Synthesis of Poly-Glutamic Acid-Camptothecin (PG-CPT)Conjugate.

[0169] The synthesis of PG-CPT followed a similar reaction as previouslydescribed for the synthesis of PG-TXL. Into 80 mg of PG polymer in 2.5ml dry DMF was added 20 mg CPT (Hande Tech.), 34 mg DCC, and traceamount of DMAP as catalyst. After stirred at room temperature overnight,the reaction mixture was poured into chloroform, and the precipitatecollected. The dried precipitate was redissolved in sodium carbonatesolution, dialyzed against 0.05 M phosphate buffer (pH4.5), filtered,and lyophilized. The content of CPT in the polymer conjugate wasdetermined by fluorescence spectrometer (Perkin-Elmer Model MPF-44A)using emission wavelength of 430 nm and excitation wavelength of 370 nm.Content: 2% to 5% (w/w), solubility: >200 mg conjugate/ml.

EXAMPLE 9 Synthesis of Poly-Lysine (PL) TXL Conjugate (PL-TXL).

[0170] All accessible amine functional groups of poly-lysine (MW>30,000,Sigma) will be converted to carboxylic acid functional groups byreacting poly-lysine with succinic anhydride, glutaric anhydride, orDTPA. The remaining unreacted NH2 group in polylysine will be blocked byreacting the modified polymer with acetic anhydride. TXL, docetaxel,other taxiods, etopside, teniposide, camptothecin, epothilone or otheranti-tumor drugs will be conjugated to the resulting polymer accordingto previously described procedures for the synthesis of PG-TXL.

EXAMPLE 10 Synthesis of Other Polyamino Acids to be Used to ConjugateTXL.

[0171] Polyamino acid copolymers containing glutamic acid may besynthesized by the copolymerization of N-carboxyanhydrides (NCAs) ofcorresponding amino acid with gamma-benzyl-L-glutamate NCA. Theresulting benzyl glutamate-containing copolymer will be converted toglutamic acid-containing copolymer by removing the benzyl protectinggroup (FIG. 15). TXL, docetaxel, other taxiods, etopside, teniposide,camptothecin, epothilone or other anti-tumor drugs will be conjugated tothe resulting polymer according to previously described procedures forthe synthesis of PG-TXL and PG-CPT.

EXAMPLE 11 Use of PG-TXL in Humans

[0172] Introduction

[0173] Poly-L-glutamic acid-Paclitaxel (PG-TXL) is a conjugate ofpoly-L-glutamic acid and paclitaxel. This compound is water soluble andbased on early animal studies it appears that it can be administered asa short, that is several minute, intravenous injection. Based on the invitro and early animal work, it appears that this compound is at leastas active against cancer as the monomeric paclitaxel in Cremophor andmay have fewer side effects. Based on these observations, this drug willbe studied in humans. The study will first require formulation of thiscompound in a solvent which is commonly used for intravenous infusion.The inventors expect that either normal saline, 5% dextrose in water orsterile water will be used as the solvent. This formulation of PG-TXLwill then be administered to at least two species of test animals suchas rats and dogs to determine the toxicities of the drug in thoseanimals and to determine a dose of the drug which then can serve as thelowest starting dose for a Phase I human study. That Phase I human studywill define a dose of PG-TXL which may be used in subsequent Phase IIstudies in patients. Phase II studies will be performed in several tumortypes to determine the activity of PG-TXL in various cancers. One ofordinary skill in the art will recognize that modifications inadministration, selection of animal models and dose regiments may bemade in the methods disclosed in following example, and suchmodifications are encompassed by the invention.

[0174] Animal Studies

[0175] These studies will be performed in rats and Beagle dogs withapproximately 3 animals studied at each dose level of the drug. Thelevels will be increased until life threatening toxicity is noted. Theanimals will undergo blood testing as well as necropsy to determine theorgan systems which are susceptible to this drug's toxicity andtherefore to expect the side effects in human studies. Once the dose isdetermined which causes the death of 10% of animals then the equivalentof one-tenth of that dose will be recommended as the starting dose forhuman studies. This is the usual recommendation by the Food and DrugAdministration (FDA) as the initial dose for human Phase I studies.

[0176] Phase I Studies

[0177] Phase I study of this drug will be performed using the startingdose defined in animal studies. The drug will be injected into the veinby a syringe over several mm or alternatively it may be infused as ashort infusion, up to approximately 10 to 15 min. The volume of thesolvent will be from 10 ml to approximately 100 ml depending on which ofthe two intravenous injection approaches are used. The drug will beadministered every 3 wk. This schedule is based on the early animalstudies and on the schema used with paclitaxel in Cremophor. Threepatients will be started on the lowest dose level as defined by theanimal studies and will be treated with an injection of PG-TXL. Bloodtests will be performed at baseline and weekly to evaluate blood counts;tests of liver function and renal function will be performed every 3 wk.It is expected that the counts and physiological parameters will recoversufficiently from the PG-TXL to resume the next cycle of treatment 3 wkafter the previous one. If this is the case then the treatment will berepeated every 3 wk. If the first cohort of three patients tolerates thedrug for 3 wk then these patients will be allowed to have the doseincreased by a predetermined schema that is usually used in the Phase Istudies. Once three patients have tolerated the first cycle, the nextcohort of 3 patients will be started on the next higher dose level. Thisprocess of increasing the dose level will continue until at least 2 outof 3 patients at a dose level have side effects which are so severe thatthey prohibit continuing administration of the drug. In such acircumstance the dose level just prior to the excessively toxic one willbe considered the level of drug to be administered in subsequentstudies. Six to ten patients shall be treated on the dose level whichwill be recommended for Phase II studies to confirm its tolerability.Once the appropriate dose has been defined and acute toxic side effectsof the drug evaluated, Phase II studies will be initiated.

[0178] Phase II Studies

[0179] Phase II studies of PG-TXL will be performed in several tumortypes. Each study will be designed in a usual standard Phase II mannerfollowing either Gahan's or Simon's design. In brief, approximately 14patients of a given tumor type will be treated initially, if there is noevidence of anti-cancer activity in that tumor type then further studiesof PG-TXL in that tumor type will be aborted. However, if at least onepatient has clinical benefit, defined as at least 50% decrease in thesum of products of perpendicular cross-sectional diameters of thetumors, then the number of patients with that tumor type treated withPG-TXL will be increased to 30. These studies will allow us to definethe activity of PG-TXL in various cancers and refine the information onthe side effects of the drug. The tumor types of special interest forPG-TXL will be the ones which have shown good response to paclitaxel anddocetaxel. This will include ovarian cancer, breast cancer, and lungcancer. Studies comparing poly-glutamic acid-paclitaxel to paclitaxel intumors showing response to PG-TXL will be performed. Such studies arecalled Phase III studies.

[0180] Phase III Studies of PG Paclitaxel

[0181] Based on the activity of paclitaxel in ovarian cancer, breastcancer, and lung cancer these will be the tumor types in which PG-TXLwill be compared to paclitaxel. In view of the necessity to have a largenumber of patients in such randomized studies, the inventors expect thata multi-institutional study will be necessary. The inventors have intheir institution access to Cooperative Community Oncology Program(CDDP) and to many other multi-institutional study groups. In additionto the potential clinical benefit of PG-TXL vs. paclitaxel, it would beappropriate to evaluate the economic impact of the two drugs. It isexpected that a short term infusion of PG-TXL may result in a lesscostly treatment. And, therefore, there is an expectation that PG-TXLmay be cost effective relative to paclitaxel monotherapy. Not only isthe infusion going to be shorter, it is expected that in view of theabsence of Cremophor fewer side effects will be experienced by thepatients and therefore the premedication regiment including steroids andintravenous H2 and Hi blockers may no longer be necessary. All of thesefactors will result in a reduction in the cost of the treatment.

[0182] Summary

[0183] It is expected that the initial animal toxicology evaluation willrequire up to 6 months. Subsequent to that, if a drug formulation isavailable, human Phase I studies may be completed in another 6 to 9months. Once these have been completed, Phase II studies in varioustumor types may take another 6 to 9 months. At that point, the inventorswill have a good idea of the efficacy of this drug and targeted PhaseIII studies may be designed and initiated. It is also possible that thePhase II studies will show enough clinical activity that abbreviatedPhase III studies or no Phase IU studies would be necessary.

EXAMPLE 12 Enhancement of Tumor Radioresponse of a Murine OvarianCarcinoma by Poly(L-Glutamic Acid)-Paclitaxel Conjugate

[0184] Introduction

[0185] The combination of chemotherapy and radiation therapy in thetreatment of a variety of tumors has produced substantial improvement incomplete response and survival rates (Rotman, 1992). Both in vitro andin vivo studies have demonstrated that paclitaxel can strongly enhancetumor radioresponse. In animal studies, the enhancement factors rangefrom 1.2 to more than 2.0, depending on the tumor type, drugconcentration, and dose scheduling. This study investigated theradiosensitization effects of poly(L-glutamic acid)-paclitaxel (PG-TXL).

[0186] Experimental Methods

[0187] PG-TXL was synthesized as described herein from poly (L-glutamicacid) (Sigma, viscosity molecular weight: 31 K). The conjugate contained20% paclitaxel (w/w) which was coupled to PG via ester linkages. FemaleC3HL'Kam mice were inoculated i.m. in the right hind leg with 5×10⁵ovarian OCa-l carcinoma cells. When tumors reached 8 mm in diameter,mice were randomly divided into 12 groups with each group consisting of6-12 mice. Mice in groups—1-5 were given saline, 14 Gy irradiationalone, or PG-TXL alone at doses of 47, 80 or 1 20 mg eg. paclitaxel/kg.Mice in groups 6-9 were given PG-TXL at 47 mg eg. Paclitaxel/kg and 14Gy local irradiation at 2, 24, 48, and 144 h after PG-TXL treatments.Group 10 was given PG-TXL at 80 mg eg paclitaxel/kg and 14 Gy at 24 hafter PG-TXL treatment. Groups 11 and 12 was given PG-TXL at 120 mg eq.Paclitaxel/kg and 14 Gy at 24 h prior or 24 h after PG-TXL treatment.PG-TXL was given in a single intravenous injection. Local gammairradiation to the tumor was delivered from a ¹³⁷Cs irradiator at a doserate of 7 Gy per minute. Tumor growth delay was determined by measuringthree orthogonal tumor diameters until tumors reached 14 mm in diameter.

[0188] Results and Discussion

[0189] The radiosensitization effects of PG-TXL were dose dependent.

[0190] At the lower PG-TXL dose of 47 mg eq. Paclitaxel/kg, asubadditive effect was observed. The mean enhancement factors variedfrom 0.54 to 0.75 depending on the timing of radiation delivery.However, a superadditive effect was observed at higher doses of PG-TXL.The mean enhancement factors increased from 0.75 to 1.8 and 4.2 whenPG-TXL was given at 24 h prior to irradiation and PG-TXL doses wereincreased from 47 to 80 and 120 mg eg. Paclitaxel/kg (Table 7). Thesubadditive effect of chemoradiation observed with PG-TXL at the lowerdose may be attributed to inadequate cell killing and rapid repopulationof surviving cells. At higher doses, PG-TXL may have profound effects onpopulation of cycling tumor cells and/or on tumor reoxygenation,resulting in significantly enhanced radiosensitization effect.Interestingly, when tumors were irradiated at 14 Gy prior to treatmentwith PG-TXL at 120 mg eq. paclitaxel/kg, a superadditive effect with anenhancement factor of 4.3 was observed (Table 7). This result contrastswith previous observation that paclitaxel induces radiation resistancewhen it was given after irradiation (Ingram and Redpath, 1997). TABLE 7Effect of PG-TXL on Radioresponse of Murine Ovarian OCa-1 Tumor Days fortumor to Absolute Normalized grow from growth growth EnhancementRadiation 8-14 mm delay in delay factors (95% Treatments (14 GY) (means± SD) days^(a) (means ± SD)^(b) C.I.)^(c) Saline No 17.2 ± 2.2 PG-TXL 47No  19.8 ± 0.98 2.7 mg eq./kg PG-TXL 80 No 25.3 ± 3.9 8.1 mg eq./kgPG-TXL 120 No 29.7 ± 3.2 12.5 mg eq/kg 14 Gy radia- Yes 37.9 ± 6.1 20.7tion alone PG-TXL 47 Yes 35.3 ± 4.7 18.2 15.5 ± 4.7 0.75 (0.5- mg eq./kg0.98) PG-TXL 80 Yes   62 ± 4.6 45.6 37.5 ± 4.6 1.8 (1.5-2.2) mg eq./kgPG-TXL 120 Yes 115 ± 3  98.4 85.9 ± 3.0 4.2 (3.9- mg eq/kg 4.3)^(e)PG-TXL 120 Yes^(d)  117 ± 1.2 100.5   88 ± 1.2 4.3 (4.1- mg eq/kg4.4)^(e)

[0191] Conclusion

[0192] The results of this study indicate that PG-TXL in combinationwith radiotherapy may be effectively used either before or afterirradiation to enhance radiosensitization. These results further suggestthat conjugation of radiosensitizers and anti-tumor drugs towater-soluble polymeric carriers may offer enhanced radiosensitizationeffect. In light of the present disclosures, one of ordinary skill inthe art will recognize that doses of PG-TXL and the other compositionsdisclosed herein, as well as the doses of radiation, either administeredexternally or internally (i.e. that is to say, radiation administered byan external radiation source, or administered systemically, for example,by injection or ingestion of radioactive materials, such as aradioisotope containing formulation), may be varied. Treatment schedulesand dosages may be varied on a patient by patient—basis, taking intoaccount, for example, factors such as the weight and age of the patient,the type of tumor being treated, the severity of the disease condition,previous and/or concurrent therapeutic interventions, the manner ofadministration and the like, which can be readily determined by one ofordinary skill in the art. For example, it is contemplated that apreferred range of doses for PG-TXL would be from about 0.5× to about 2×the maximum tolerated dose of TXL in equivalent paclitaxel doses. Theamount of PG-TXL administered may be spread over the course of radiationtherapy as sub-doses. It is also contemplated that a preferred range ofirradiation would be about 50 to about 70 Gray (Gy) administered overabout 5 to about 7 weeks or about 10 Gray per week. Preferred schedulesof administration would include administering PG-TXL about 1 to about 2days before, or about 1 to about 2 days after irradiation. Schedules ofadministration of PG-TXL and other polymer-antitumor drug orchelator-antitumor drug compositions, of course, may be varied andlorrepeated as determined by one of ordinary skill in the art for themaximum benefit of each patient.

[0193] While the compositions and methods of this invention have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions, methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related, asother water soluble polymer-drug conjugates may be substituted for theagents described herein, the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

References

[0194] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0195] Bartoni and Boitard, “In vitro and in vivo antitumoral activityof free, and encapsulated taxol,” J. Microencapsulation, 7:191-197,1990.

[0196] Berstein, et al., “Higher antitumor efficacy of daunomycin whenlinked to dextran: In vitro and in vivo studies,” J. Natl. Cancer Inst.,60:379-384, 1978,

[0197] Boyle, et al, “Prevention of taxol induced neuropathy byglutamate,” Cancer Res., 37:290. 1996.

[0198] Cabral and Barlow, “Resistance to antimitotic agents as geneticprobes of microtubule structure and function,” Pharmac. Ther.,52:159-171, 1991.

[0199] Cabral, Wible, Brenner, Brinkley, “Taxol-requiring mutant ofChinese hamster ovary cells with impaired mitotic spindle assembly,” J.Cell Biol., 97:30-39, 1983.

[0200] Cortes, J. E. and Pazdur, R., “Docetaxel,” Journal of ClinicalOncology 13:2643-2655, 1995.

[0201] Deutsch et al., “Synthesis of congeners and prodrugs. 3.Water-soluble prodrugs of paclitaxel with potent antitumor activity,” J.Med. Chem., 32:788-792, 1989.

[0202] Duncan, et al., “Anticancer agents coupled toN-(2hydroxypropyl)methacryamide copolymers. 3. Evaluation of adriamycinconjugates against mouse leukemia L1210 in vivo,” J. Controlled Rel.,10:51-63, 1989.

[0203] Eiseman et al., “Plasma pharmacokinetics and tissue distributionof paclitaxel in CD2F1 mice,” Cancer Chemother. Pharmacol., 34:465-471,1994.

[0204] Fidler, Gersten, Hart, “The biology of cancer invasion andmetastasis,” Adv. Cancer Res., 28:149-250, 1987.

[0205] Foa, Norton, Seidman, “Taxol (paclitaxel): a novelanti-microtubule agent with remarkable anti-neoplastic activity,” Int.J. Clin. Lab. Res., 24:6-14, 1994.

[0206] Goldspiel, “Taxol pharmaceutical issues: preparation,administration, stability, and compatibility with other medications,”Ann. Pharmacotherapy, 28:S23-26, 1994.

[0207] Greenwald et al., “Highly water soluble taxol derivative:2′-polyethylene glycol esters as potential products,” Bioorganic &Medicinal Chemistry Letters, 4:2465-2470, 1994.

[0208] Greenwald et al., “Highly water soluble Taxol derivatives,7-polyethylene glycol esters as potential products,” J. Org. Chem.,60:331-336, 1995.

[0209] Greenwald, et al., “Drug delivery systems: Water soluble taxol2′-poly(ethylene glycol) ester prodrugs-design and in vivoeffectiveness,” J. Med. Chem., 39:424-431, 1996.

[0210] Hirano et al., “Polymeric derivatives of activatedcyclophosphamide as drug delivery systems in antitumor therapy,”Macromol. Chem., 180:1125-1130, 1979.

[0211] Hoes et al., “Optimization of macromolecular prodrugs of theantitumor antibiotic adriamycin,” J. Controlled Release, 2:205-213,1985.

[0212] Holmes, Kudelka, Kavanagh, Huber, Ajani, Valero, “Current statusof clinical trials with paclitaxel and docetaxel,” In: Taxane AnticancerAgents: Basic Science and Current Status, Georg, Chen, Ojima, Vyas,eds., American Chemical Society, Washington, DC, 31-57, 1995.

[0213] Horwitz et al., “Taxol, mechanisms of action and resistance,” J.Natl. Cancer Inst. Monographs No. 15, pp. 55-61, 1993.

[0214] Kato, et al., “Antitumor activity of 1 -barabinofuranosylcytosineconjugated with polyglutamic acid and its derivative,” Cancer Res.,44:25-30, 1984.

[0215] Kopecek and Kopeckova. “Targetable water-soluble polymericanticancer drugs: achievements and unsolved problems,” Proceed. InternSymp. Control. Rel. Bioact. Mater., 20:190-191, 1993.

[0216] Kopecek, “The potential of water-soluble polymeric carriers intargeted and site-specific drug delivery,” J. Controlled Release,11:279-290, 1990.

[0217] Li, et al., “Synthesis and evaluation of water-solublepolyethylene glycol paclitaxel conjugate as a paclitaxel prodrug,”Anti-Cancer Drugs, 7:642-648, 1996.

[0218] Li, Yu, Newman, Cabral, Stephens, Hunter, Milas, Wallace,“Complete regression of well-established tumors using a novelwater-soluble poly(L-glutamic acid)paclitaxel conjugate,” Cancer Res.,58:2404-2409, 1998.

[0219] Liu, et al., “Evidence for involvement of tyrosinephosphorylation in taxol-induced apoptosis in a human ovarian tumor cellline,” Biochem. Pharmacol., 48:1265-1272, 1994.

[0220] Longnecker, et al., “High performance liquid chromatographicassay for Taxol in human plasma and urine and pharmacokinetics in aphase I trial,” Cancer Treat. Rep., 71:53-59, 1987.

[0221] Maeda and Matsumura, “Tumoritropic and lymphotropic principles ofmacromolecular drugs,” Critical Review in Therapeutic Drug CarrierSystems, 6:193-210, 1989.

[0222] Maeda, “SMANCS and polymer-conjugated macromolecular drugs:advantages in cancer chemotherapy,” Adv. Drug Delivery Rev., 6:181-193,1991.

[0223] Maeda, Seymour, Miyamoto, “Conjugates of anticancer agents andpolymers: Advantages of macromolecular therapeutics in vivo,” Bioconjug.Chem., 3:351-362, 1992.

[0224] Magri and Kingston, “Modified taxols. 2. Oxidation products oftaxol,” J. Org. Chem., 51:797-802, 1986.

[0225] Mathew et al., “Synthesis and evaluation of some water-solubleprodrugs and derivatives of taxol with antitumor activity,” J. Med.Chem., 35:145-151, 1992.

[0226] Milas, et al., “Kinetics of mitotic arrest and apoptosis inmurine mammary and ovarian tumors treated with taxol,” Proc. Am. Assoc.Cancer Chemother. Pharmacol., 35:297-303, 1995.

[0227] Mosmann, T., “Rapid colormetric assay for cellular growth andsurvival: application to proliferation and cytotoxic assay,” J. Immunol.Methods, 65:55-63, 1983.

[0228] Nicolaou, Riemer, Kerr, Rideout, Wrasidio, “Design, synthesis andbiological activity of protaxols,” Nature, 364:464-466, 1993.

[0229] Oliver, S. J. et al., Suppression of collagen-induced arthritisusing an angiogenesis inhibitor, AGM-1470, and a microtubule stabilizer,Taxol,” Cellular Immunology 157:291-299, 1994.

[0230] Phillips-Hughes and Kandarpa, “Restenosis: pathophysiology andpreventive strategies,” JVIR 7:321-333, 1996.

[0231] Reynolds, T., “Polymers help guide cancer drugs to tumor targets-and keep them there,” J. Natl. Cancer Institute, 87:1582-1584, 1995.

[0232] Rose, et al., “Preclinical antitumor activity of water-solublepaclitaxel derivatives,” Cancer Chemother. Pharmacol., 39:486-492, 1997.

[0233] Rotman, “Chemoirradiation: a new initiative in cancer treatment.1991 RSNA annual oration in radiation oncology,” Radiology, 184:319-329,1992.

[0234] Rowinsky and Donehower, “Review: Paclitaxel (Taxol),” N. Engl. J.Med., 332:1004-1014, 1995.

[0235] Rowinsky, Chaudhry, Cornblath, Donehower, “Phase I andpharmacologic study of paclitaxel and cisplatin with granulocytecolony-stimulating factor: neuromuscular toxicity is dose-limiting,” J.Clin. Oncol., 11:2010-2020, 1993.

[0236] Scudiero et al. “Evaluation of a Soluble Tetrazolium/FormazanAssay for Cell Growth and Drug Sensitivity in Culture Using Human andOther Tumor Cell Lines,” Cancer Research, 48:4827-4833, 1988.

[0237] Serruys, De Jaegere, Kiemeneij et al., “A comparison ofballoon-expandable-stent implantation with balloon angioplasty inpatients with coronary artery disease,” N. Engl. J. Med., 331:489-495,1994.

[0238] Sharma and Straubinger, “Novel taxol formulations: Preparationand Characterization of taxol-containing liposomes,” Pharm. Res.11:889-896, 1994.

[0239] Trouet, et al., “A covalent linkage between daunorubicin andproteins that is stable in serum and reversible by lysosomal hydrolases,as required for a lysosomotropic drug-carrier conjugate: In vitro and invivo studies,” Proc. Natl. Acad. Sci. U.S.A., 79:626-629, 1982.

[0240] U.S. Pat. No. 5,583,153

[0241] U.S. Pat. No. 5,362,831

[0242] van Heeswijk et al., “The synthesis and characterization ofpolypeptide-adriamycin conjugate and its complexes with adriamycin. Part1,” J. Controlled Release, 1:301-315, 1985.

[0243] Vyas et al., “Phosphatase-activated prodrugs of paclitaxel,”In:Taxane Anticancer Agents: Basic Science and Current Status, Georg,Chen, Ojima, Vyas. eds., American Chemical Society, Washington, DC,124-137, 1995.

[0244] Weiss et al., “Hypersensitivity reactions from Taxol,” J. Clin.Oncol., 8:1263-1268, 1990.

[0245] Zhao, Z. and Kingston, D. G. I., “Modified taxols. 6. Preparationof water-soluble taxol phosphates,” J. Nat. Prod., 54:1607-1611, 1991.

What is claimed is:
 1. A method of enhancing the response of a tumor toirradiation, comprising: a) administering to a patient in need of suchtherapy a radiosensitizing amount of a pharmaceutical compositioncomprising paclitaxel, docetaxel, eptopside, teniposide, camptothecin orepothilone conjugated to a water soluble polyamino acid polymer and apharmaceutically acceptable carrier; b) irradiating said tumor; whereinsaid conjugated paclitaxel or docetaxel have increased water solubility,efficacy and accumulation within a tumor compared with the correspondingunconjugated drugs.
 2. The method of claim 1, wherein said polymer isselected form polyglutamic acids, polyaspartic acids or polylysines. 3.The method of claim 2, wherein said polymer has a molecular weight ofabout 5000 to about 100000 daltons.
 4. The method of claim 2, whereinsaid polymer has a molecular weight of about 20000 to about 80000daltons.
 5. The method of claim 2, wherein said polymer has a molecularweight of about 25000 to about 50000 daltons.
 6. The method of claim 1,wherein said polymer is a polyglutamic acid.
 7. The method of claim 6,wherein said conjugate comprises from about 2% to about 35% by weight ofpaclitaxel or docetaxel.
 8. The method of claim 1, wherein step (b) iscarried out by administering gamma irradiation to said tumor.
 9. Themethod of claim 9, wherein said composition is administered prior toirradiation.
 10. The method of claim 9, wherein said composition isadministered following irradiation.
 11. The method of claim 9, whereinsaid irradiation dose is about 10 Gray per week, and said composition isadministered within 1-2 days of irradiation.
 12. The method of claim 9,wherein said cancer is breast cancer, ovarian cancer, malignantmelanoma, lung cancer, gastric cancer, prostate cancer, colon cancer,head and neck cancer; leukemia or Kaposi's sarcoma.
 13. A method oftreating cancer comprising: a) administering to a patient in need ofsuch therapy a radiosensitizing amount of a pharmaceutical compositioncomprising paclitaxel, docetaxel, eptopside, teniposide, camptothecin orepothilone conjugated to a polyglutamic acid polymer and apharmaceutically acceptable carrier; and b) irradiating said tumor. 14.The method of claim 13, wherein the amount of said composition is fromabout 0.5 times to about 2 times the maximum tolerated doses ofpaclitaxel in equivalent paclitaxel doses.